Method of preparing gas and gaseous precursor-filled microspheres

ABSTRACT

Methods of and apparatus for preparing temperature activated gaseous precursor-filled liposomes are described. Gaseous precursor-filled liposomes prepared by these methods are particularly useful, for example, in ultrasonic imaging applications and in therapeutic drug delivery systems.

RELATED APPLICATIONS

This application is continuation-in-part of applications U.S. Ser. Nos.08/160,232 and 08/159,674, now abandoned, filed concurrently herewith onNov. 30, 1993, which are continuations-in-part of application U.S. Ser.No. 076,239, filed Jun. 11, 1993, now U.S. Pat. No. 5,469,854 which is acontinuation-in-part of application U.S. Ser. No. 717,084, now U.S. Pat.No. 5,228,446 and U.S. Ser. No. 716,899, now abandoned, both of whichwere filed Jun. 18, 1991, which in turn are a continuation-in-part ofU.S. Ser. No. 569,828, filed Aug. 20, 1990, now U.S. Pat. No. 5,088,499which in turn is a continuation-in-part of application U.S. Ser. No.455,707, filed Dec. 22, 1989, which is abandoned. The disclosures ofeach of these patent applications are incorporated herein by referencein their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to novel methods and apparatus for preparinggaseous precursor-filled liposomes. Liposomes prepared by these methodsare particularly useful, for example, in ultrasonic imaging applicationsand in therapeutic delivery systems.

2. Background of the Invention

A variety of imaging techniques have been used to detect and diagnosedisease in animals and humans. X-rays represent one of the firsttechniques used for diagnostic imaging. The images obtained through thistechnique reflect the electron density of the object being imaged.Contrast agents such as barium or iodine have been used over the yearsto attenuate or block X-rays such that the contrast between variousstructures is increased. X-rays, however, are known to be somewhatdangerous, since the radiation employed in X-rays is ionizing, and thevarious deleterious effects of ionizing radiation are cumulative.

Another important imaging technique is magnetic resonance imaging (MRI).This technique, however, has various drawbacks such as expense and thefact that it cannot be conducted as a portable examination. In addition,MRI is not available at many medical centers.

Radionuclides, employed in nuclear medicine, provide a further imagingtechnique. In employing this technique, radionuclides such as technetiumlabelled compounds are injected into the patient, and images areobtained from gamma cameras. Nuclear medicine techniques, however,suffer from poor spatial resolution and expose the animal or patient tothe deleterious effects of radiation. Furthermore, the handling anddisposal of radionuclides is problematic.

Ultrasound is another diagnostic imaging technique which is unlikenuclear medicine and X-rays since it does not expose the patient to theharmful effects of ionizing radiation. Moreover, unlike magneticresonance imaging, ultrasound is relatively inexpensive and can beconducted as a portable examination. In using the ultrasound technique,sound is transmitted into a patient or animal via a transducer. When thesound waves propagate through the body, they encounter interfaces fromtissues and fluids. Depending on the acoustic properties of the tissuesand fluids in the body, the ultrasound sound waves are partially orwholly reflected or absorbed. When sound waves are reflected by aninterface they are detected by the receiver in the transducer andprocessed to form an image. The acoustic properties of the tissues andfluids within the body determine the contrast which appears in theresultant image.

Advances have been made in recent years in ultrasound technology.However, despite these various technological improvements, ultrasound isstill an imperfect tool in a number of respects, particularly withregard to the imaging and detection of disease in the liver and spleen,kidneys, heart and vasculature, including measuring blood flow. Theability to detect and measure these regions depends on the difference inacoustic properties between tissues or fluids and the surroundingtissues or fluids. As a result, contrast agents have been sought whichwill increase the acoustic difference between tissues or fluids and thesurrounding tissues or fluids in order to improve ultrasonic imaging anddisease detection.

The principles underlying image formation in ultrasound have directedresearchers to the pursuit of gaseous contrast agents. Changes inacoustic properties or acoustic impedance are most pronounced atinterfaces of different substances with greatly differing density oracoustic impedance, particularly at the interface between solids,liquids and gases. When ultrasound sound waves encounter suchinterfaces, the changes in acoustic impedance result in a more intensereflection of sound waves and a more intense signal in the ultrasoundimage. An additional factor affecting the efficiency or reflection ofsound is the elasticity of the reflecting interface. The greater theelasticity of this interface, the more efficient the reflection ofsound. Substances such as gas bubbles present highly elastic interfaces.Thus, as a result of the foregoing principles, researchers have focusedon the development of ultrasound contrast agents based on gas bubbles orgas containing bodies and on the development of efficient methods fortheir preparation.

Ryan et al., in U.S. Pat. No. 4,544,545, disclose phospholipid liposomeshaving a chemically modified cholesterol coating. The cholesterolcoating may be a monolayer or bilayer. An aqueous medium, containing atracer, therapeutic, or cytotoxic agent, is confined within theliposome. Liposomes, having a diameter of 0.001 microns to 10 microns,are prepared by agitation and ultrasonic vibration.

D'Arrigo, in U.S. Pat. Nos. 4,684,479 and 5,215,680, teaches agas-in-liquid emulsion and method for the production thereof fromsurfactant mixtures. U.S. Pat. No. 4,684,479 discloses the production ofliposomes by shaking a solution of the surfactant in a liquid medium inair. U.S. Pat. No. 5,215,680 is directed to a large scale method ofproducing lipid coated microbubbles including shaking a solution of thesurfactant in liquid medium in air or other gaseous mixture and filtersterilizing the resultant solution.

WO 80/02365 discloses the production of microbubbles having an inertgas, such as nitrogen; or carbon dioxide, encapsulated in a gellablemembrane. The liposomes may be stored at low temperatures and warmedprior and during use in humans. WO 82/01642 describes microbubbleprecursors and methods for their production. The microbubbles are formedin a liquid by dissolving a solid material. Gas-filled voids result,wherein the gas is 1.) produced from gas present in voids between themicroparticles of solid precursor aggregates, 2.) absorbed on thesurfaces of particles of the precursor, 3.) an integral part of theinternal structure of particles of the precursor, 4.) formed when theprecursor reacts chemically with the liquid, and 5.) dissolved in theliquid and released when the precursor is dissolved therein.

In addition, Feinstein, in U.S. Pat. Nos. 4,718,433 and 4,774,958,teaches the use of albumin coated microbubbles for the purposes ofultrasound.

Widder, in U.S. Pat. Nos. 4,572,203 and 4,844,882, discloses a method ofultrasonic imaging and a microbubble-type ultrasonic imaging agent.

Quay, in WO 93/05819, describes the use of agents to form microbubblescomprising especially selected gases based upon a criteria of knownphysical constants, including 1) size of the bubble, 2) density of thegas, 3) solubility of the gas in the surrounding medium, and 4)diffusivity of the gas into the medium.

Kaufman et al., in U.S. Pat. No. 5,171,755, disclose an emulsioncomprising an highly fluorinated organic compound, an oil having nosubstantial surface activity or water solubility and a surfactant.Kaufman et al. also teach a method of using the emulsion in medicalapplications.

Another area of significant research effort is in the area of targeteddrug delivery. Targeted delivery means are particularly important wheretoxicity is an issue. Specific therapeutic delivery methods potentiallyserve to minimize toxic side effects, lower the required dosage amounts,and decrease costs for the patient.

The methods and materials in the prior art for introduction of geneticmaterials, for example, to living cells is limited and ineffective. Todate several different mechanisms have been developed to deliver geneticmaterial to living cells. These mechanisms include techniques such ascalcium phosphate precipitation and electroporation, and carriers suchas cationic polymers and aqueous-filled liposomes. These methods haveall been relatively ineffective in vivo and only of limited use for cellculture transfection. None of these methods potentiate local release,delivery and integration of genetic material to the target cell.

Better means of delivery for therapeutics such as genetic materials areneeded to treat a wide variety of human and animal diseases. Greatstrides have been made in characterizing genetic diseases and inunderstanding protein transcription but relatively little progress hasbeen made in delivering genetic material to cells for treatment of humanand animal disease.

A principal difficulty has been to deliver the genetic material from theextracellular space to the intracellular space or even to effectivelylocalize genetic material at the surface of selected cell membranes. Avariety of techniques have been tried in vivo but without great success.For example, viruses such as adenoviruses and retroviruses have beenused as vectors to transfer genetic material to cells. Whole virus hasbeen used but the amount of genetic material that can be placed insideof the viral capsule is limited and there is concern about possibledangerous interactions that might be caused by live virus. The essentialcomponents of the viral capsule may be isolated and used to carrygenetic material to selected cells. In vivo, however, not only must thedelivery vehicle recognize certain cells but it also must be deliveredto these cells. Despite extensive work on viral vectors, it has beendifficult to develop a successfully targeted viral mediated vector fordelivery of genetic material in vivo.

Conventional, liquid-containing liposomes have been used to delivergenetic material to cells in cell culture but have mainly beenineffective in vivo for cellular delivery of genetic material. Forexample, cationic liposome transfection techniques have not workedeffectively in vivo. More effective means are needed to improve thecellular delivery of therapeutics such as genetic material.

The present invention is directed to addressing the foregoing, as wellas other important needs in the area of contrast agents for ultrasonicimaging and vehicles for the effective targeted delivery oftherapeutics.

SUMMARY OF THE INVENTION

The present invention provides methods and apparatus for preparingtemperature activated gaseous precursor-filled liposomes suitable foruse as contrast agents for ultrasonic imaging or as drug deliveryagents. The methods of the present invention provide the advantages, forexample, of simplicity and potential cost savings during manufacturingof temperature activated gaseous precursor-filled liposomes.

Preferred methods for preparing the temperature activated gaseousprecursor-filled liposomes comprise shaking an aqueous solutioncomprising a lipid in the presence of a temperature activated gaseousprecursor, at a temperature below the gel state to liquid crystallinestate phase transition temperature of the lipid.

Unexpectedly, the temperature activated gaseous precursor-filledliposomes prepared in accordance with the methods of the presentinvention possess a number of surprising yet highly beneficialcharacteristics. For example, gaseous precursor-filled liposomes areadvantageous due to their biocompatibility and the ease with whichlipophilic compounds can be made to cross cell membranes after theliposomes are ruptured. The liposomes of the invention also exhibitintense echogenicity on ultrasound, are highly stable to pressure,and/or generally possess a long storage life, either when stored dry orsuspended in a liquid medium. The echogenicity of the liposomes is ofimportance to the diagnostic and therapeutic applications of theliposomes made according to the invention. The gaseous precursor-filledliposomes also have the advantages, for example, of stable particlesize, low toxicity and compliant membranes. It is believed that theflexible membranes of the gaseous precursor-filled liposomes may beuseful in aiding the accumulation or targeting of these liposomes totissues such as tumors.

The temperature activated gaseous precursor-filled liposomes madeaccording to the present invention thus have superior characteristicsfor ultrasound contrast imaging. When inside an aqueous or tissue media,the gaseous precursor-filled liposome creates an interface for theenhanced absorption of sound. The gaseous precursor-filled liposomes aretherefore useful in imaging a patient generally, and/or in diagnosingthe presence of diseased tissue in a patient as well as in tissueheating and the facilitation of drug release or activation.

In addition to ultrasound, the temperature activated gaseousprecursor-filled liposomes made according to the present invention maybe used, for example, for magnetic imaging and as MRI contrast agents.For example, the gaseous precursor-filled liposomes may containparamagnetic gases, such as atmospheric air, which contains traces ofoxygen 17; paramagnetic ions such as Mn⁺², Gd⁺², Fe⁺³ ; iron oxides; ormagnetite (Fe₃ O₄) and may thus be used as susceptibility contrastagents for magnetic resonance imaging. Additionally, for example, thegaseous precursor-filled liposomes made according to the presentinvention may contain radioopaque metal ions, such as iodine, barium,bromine, or tungsten, for use as x-ray contrast agents.

The temperature activated gaseous precursor-filled liposomes are alsoparticularly useful as drug carriers. Unlike liposomes of the prior artthat have a liquid interior suitable only for encapsulating drugs thatare water soluble, the gaseous precursor-filled liposomes made accordingto the present invention are particularly useful for encapsulatinglipophilic drugs. Furthermore, lipophilic derivatives of drugs may beincorporated into the lipid layer readily, such as alkylated derivativesof metallocene dihalides. Kuo et al., J. Am. Chem. Soc. 1991, 113,9027-9045.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a view, partially schematic, of a preferred apparatusaccording to the present invention for preparing the gaseousprecursor-filled liposome microspheres of the present invention.

FIG. 2 shows a preferred apparatus for filtering and/or dispensingtherapeutic containing gaseous precursor-filled liposome microspheres ofthe present invention.

FIG. 3 shows a preferred apparatus for filtering and/or dispensingtherapeutic containing gaseous precursor-filled liposome microspheres ofthe present invention.

FIG. 4 is an exploded view of a portion of the apparatus of FIG. 3.

FIGS. 5A and B are micrographs which show the sizes of gaseousprecursor-filled liposomes of the invention before (A) and after (B)filtration.

FIGS. 6A and B graphically depict the size distribution of gaseousprecursor-filled liposomes of the invention before (A) and after (B)filtration.

FIGS. 7A and B are micrographs of a lipid suspension before (A) andafter (B) extrusion through a filter.

FIGS. 8A and B are micrographs of gaseous precursor-filled liposomesformed subsequent to filtering and autoclaving a lipid suspension, themicrographs having been taken before (A) and after (B) sizing byfiltration of the gaseous precursor-filled liposomes.

FIG. 9 is a diagrammatic illustration of a temperature activated gaseousprecursor-filled liposome prior to temperature activation. The liposomehas a multilamellar membrane.

FIG. 10 is a diagrammatic illustration of a temperature activated liquidgaseous precursor-filled liposome after temperature activation of theliquid to gaseous state resulting in a unilamellar membrane andexpansion of the liposome diameter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to methods and apparatus for preparingtemperature activated gaseous precursor-filled liposomes. Unlike themethods of the prior art which are directed to the formation ofliposomes with an aqueous solution filling the interior, the methods ofthe present invention are directed to the preparation of liposomes whichcomprise interior gaseous precursor and/or ultimately gas.

As used herein, the phrase "temperature activated gaseous precursor"denotes a compound which, at a selected activation or transitiontemperature, changes phases from a liquid to a gas. Activation ortransition temperature, and like terms, refer to the boiling point ofthe gaseous precursor, the temperature at which the liquid to gaseousphase transition of the gaseous precursor takes place. Useful gaseousprecursors are those gases which have boiling points in the range ofabout -100° C. to 70° C. The activation temperature is particular toeach gaseous precursor. This concept is illustrated in FIGS. 9 and 10.An activation temperature of about 37° C., or human body temperature, ispreferred for gaseous precursors of the present invention. Thus, aliquid gaseous precursor is activated to become a gas at 37° C. However,the gaseous precursor may be in liquid or gaseous phase for use in themethods of the present invention. The methods of the present inventionmay be carried out below the boiling point of the gaseous precursor suchthat a liquid is incorporated into a microsphere. In addition, themethods may be performed at the boiling point of the gaseous precursorsuch that a gas is incorporated into a microsphere. For gaseousprecursors having low temperature boiling points, liquid precursors maybe emulsified using a microfluidizer device chilled to a lowtemperature. The boiling points may also be depressed using solvents inliquid media to utilize a precursor in liquid form. Alternatively, anupper limit of about 70° C. may be attained with focused high energyultrasound. Further, the methods may be performed where the temperatureis increased throughout the process, whereby the process starts with agaseous precursor as a liquid and ends with a gas.

The gaseous precursor may be selected so as to form the gas in situ inthe targeted tissue or fluid, in vivo upon entering the patient oranimal, prior to use, during storage, or during manufacture. The methodsof producing the temperature-activated gaseous precursor-filledmicrospheres may be carried out at a temperature below the boiling pointof the gaseous precursor. In this embodiment, the gaseous precursor isentrapped within a microsphere such that the phase transition does notoccur during manufacture. Instead, the gaseous precursor-filledmicrospheres are manufactured in the liquid phase of the gaseousprecursor. Activation of the phase transition may take place at any timeas the temperature is allowed to exceed the boiling point of theprecursor. Also, knowing the amount of liquid in a droplet of liquidgaseous precursor, the size of the liposomes upon attaining the gaseousstate may be determined.

Alternatively, the gaseous precursors may be utilized to create stablegas-filled microspheres which are pre-formed prior to use. In thisembodiment, the gaseous precursor is added to a container housing asuspending and/or stabilizing medium at a temperature below theliquid-gaseous phase transition temperature of the respective gaseousprecursor. As the temperature is then exceeded, and an emulsion isformed between the gaseous precursor and liquid solution, the gaseousprecursor undergoes transition from the liquid to the gaseous state. Asa result of this heating and gas formation, the gas displaces the air inthe head space above the liquid suspension so as to form gas-filledlipid spheres which entrap the gas of the gaseous precursor, ambient gas(e.g. air) or coentrap gas state gaseous precursor and ambient air. Thisphase transition can be used for optimal mixing and stabilization of thecontrast medium. For example, the gaseous precursor, perfluorobutane,can be entrapped in liposomes and as the temperature is raised, beyond3° C. (boiling point of perfluorobutane) liposomally entrappedfluorobutane gas results. As an additional example, the gaseousprecursor fluorobutane, can be suspended in an aqueous suspensioncontaining emulsifying and stabilizing agents such as glycerol orpropylene glycol and vortexed on a commercial vortexer. Vortexing iscommenced at a temperature low enough that the gaseous precursor isliquid and is continued as the temperature of the sample is raised pastthe phase transition temperature from the liquid to gaseous state. In sodoing, the precursor converts to the gaseous state during themicroemulsification process. In the presence of the appropriatestabilizing agents, surprisingly stable gas-filled liposomes result.

Accordingly, the gaseous precursors of the present invention may beselected to form a gas-filled liposome in vivo or designed to producethe gas-filled liposome in situ, during the manufacturing process, onstorage, or at some time prior to use.

As a further embodiment of this invention, by preforming the liquidstate of the gaseous precursor into an aqueous emulsion and maintaininga known size, the maximum size of the microbubble may be estimated byusing the idea gas law, once the transition to the gaseous state iseffectuated. For the purpose of making gaseous microspheres from gaseousprecursors, the gas phase is assumed to form instantaneously and no gasin the newly formed microbubble has been depleted due to diffusion intothe liquid (generally aqueous in nature). Hence, from a known liquidvolume in the emulsion, one actually would predict an upper limit to thesize of the gaseous liposome.

Pursuant to the present invention, an emulsion of lipid gaseousprecursor-containing liquid droplets of defined size may be formulated,such that upon reaching a specific temperature, the boiling point of thegaseous precursor, the droplets will expand into gas liposomes ofdefined size. The defined size represents an upper limit to the actualsize because factors such as gas diffusing into solution, loss of gas tothe atmosphere, and the effects of increased pressure are factors forwhich the ideal gas law cannot account.

The ideal gas law and the equation for calculating the increase involume of the gas bubbles on transition from the liquid to gaseousstates follows:

The ideal gas law predicts the following:

    PV=nRT

where

P=pressure in atmospheres

V=volume in liters

n=moles of gas

T=temperature in ° K.

R=ideal gas constant=22.4 L atmospheres deg⁻¹ mole⁻¹

With knowledge of volume, density, and temperature of the liquid in theemulsion of liquids, the amount (e.g. number of moles) of liquidprecursor as well as the volume of liquid precursor, a priori, may becalculated, which when converted to a gas, will expand into a liposomeof known volume. The calculated volume will reflect an upper limit tothe size of the gaseous liposome assuming instantaneous expansion into agas liposome and negligible diffusion of the gas over the time of theexpansion.

Thus, stabilization of the precursor in the liquid state in an emulsionwhereby the precursor droplet is spherical, the volume of the precursordroplet may be determined by the equation:

    Volume (sphere)=4/3πr.sup.3

where

r=radius of the sphere

Thus, once the volume is predicted, and knowing the density of theliquid at the desired temperature, the amount of liquid (gaseousprecursor) in the droplet may be determined. In more descriptive terms,the following can be applied:

    V.sub.gas =4/3 π(r.sub.gas).sup.3

by the ideal gas law,

    PV=nRT

substituting reveals,

    V.sub.gas =nRT/P.sub.gas

or,

(A) n=4/3[πr_(gas) ³ ] P/RT

amount n=4/3[πr_(gas) ³ P/RT] * MW_(n)

Converting back to a liquid volume

(B) V_(Liq) =[4/3[πr_(gas) ³ ] P/RT] * MW_(n) /D]

where D=the density of the precursor

Solving for the diameter of the liquid droplet,

(C) diameter/2=[3/4π [4/3 * [πr_(gas) ³ ] P/RT] MW_(n) /D]^(1/3)

which reduces to

Diameter=2[[r_(gas) ³ ] P/RT [MW_(n) /D]]^(1/3)

As a further embodiment of the present invention, with the knowledge ofthe volume and especially the radius, the appropriately sized filtersizes the gaseous precursor droplets to the appropriate diameter sphere.

A representative gaseous precursor may be used to form a microsphere ofdefined size, for example, 10 microns diameter. In this example, themicrosphere is formed in the bloodstream of a human being, thus thetypical temperature would be 37° C. or 310° K. At a pressure of 1atmosphere and using the equation in (A), 7.54×10⁻¹⁷ moles of gaseousprecursor would be required to fill the volume of a 10 micron diametermicrosphere.

Using the above calculated amount of gaseous precursor, and1-fluorobutane, which possesses a molecular weight of 76.11, a boilingpoint of 32.5° C. and a density of 6.7789 grams/mL⁻¹ at 20° C., furthercalculations predict that 5.74×10⁻¹⁵ grams of this precursor would berequired for a 10 micron microsphere. Extrapolating further, and armedwith the knowledge of the density, equation (B) further predicts that8.47×10⁻¹⁶ mLs of liquid precursor are necessary to form a microspherewith an upper limit of 10 microns.

Finally, using equation (C), an emulsion of lipid droplets with a radiusof 0.0272 microns or a corresponding diameter of 0.0544 microns need beformed to make a gaseous precursor filled microsphere with an upperlimit of a 10 micron microsphere.

An emulsion of this particular size could be easily achieved by the useof an appropriately sized filter. In addition, as seen by the size ofthe filter necessary to form gaseous precursor droplets of defined size,the size of the filter would also suffice to remove any possiblebacterial contaminants and, hence, can be used as a sterile filtrationas well.

This embodiment of the present invention may be applied to all gaseousprecursors activated by temperature. In fact, depression of the freezingpoint of the solvent system allows the use gaseous precursors whichwould undergo liquid-to-gas phase transitions at temperatures below 0°C. The solvent system can be selected to provide a medium for suspensionof the gaseous precursor. For example, 20% propylene glycol miscible inbuffered saline exhibits a freezing point depression well below thefreezing point of water alone. By increasing the amount of propyleneglycol or adding materials such as sodium chloride, the freezing pointcan be depressed even further.

The selection of appropriate solvent systems may be explained byphysical methods as well. When substances, solid or liquid, hereinreferred to as solutes, are dissolved in a solvent, such as water basedbuffers for example, the freezing point is lowered by an amount that isdependent upon the composition of the solution. Thus, as defined byWall, one can express the freezing point depression of the solvent bythe following:

    Inx.sub.a =In(1-X.sub.b)=ΔH.sub.fus /R(1/T.sub.o -1/T)

where:

x_(a) =mole fraction of the solvent

x_(b) =mole fraction of the solute

ΔH_(fus) =heat of fusion of the solvent

T_(o) =Normal freezing point of the solvent

The normal freezing point of the solvent results. If x_(b) is smallrelative to x_(a), then the above equation may be rewritten:

    X.sup.b =ΔH.sub.fus /R[T-T.sub.o /T.sub.o T]˜ΔH.sub.fus ΔT/RT.sub.o.sup.2

The above equation assumes the change in temperature ΔT is smallcompared to T₂. The above equation can be simplified further assumingthe concentration of the solute (in moles per thousand grams of solvent)can be expressed in terms of the molality, m. Thus,

    X.sub.b =m/[m+1000/Ma]˜mMa/1000

where:

Ma=Molecular weight of the solvent, and

m=molality of the solute in moles per 1000 grams.

Thus, substituting for the fraction x_(b) :

    ΔT=[M.sub.a RT.sub.o.sup.2 /1000ΔH.sub.fus ]m

or

    ΔT=K.sub.f m,

where

    K.sub.f =M.sub.a RT.sub.o.sup.2 /1000ΔH.sub.fus

K_(f) is referred to as the molal freezing point and is equal to 1.86degrees per unit of molal concentration for water at one atmospherepressure. The above equation may be used to accurately determine themolal freezing point of gaseous-precursor filled microsphere solutionsof the present invention.

Hence, the above equation can be applied to estimate freezing pointdepressions and to determine the appropriate concentrations of liquid orsolid solute necessary to depress the solvent freezing temperature to anappropriate value.

Methods of preparing the temperature activated gaseous precursor-filledliposomes include:

vortexing an aqueous suspension of gaseous precursor-filled liposomes ofthe present invention; variations on this method include optionallyautoclaving before shaking, optionally heating an aqueous suspension ofgaseous precursor and lipid, optionally venting the vessel containingthe suspension, optionally shaking or permitting the gaseous precursorliposomes to form spontaneously and cooling down the gaseous precursorfilled liposome suspension, and optionally extruding an aqueoussuspension of gaseous precursor and lipid through a filter of about 0.22μm, alternatively, filtering may be performed during in vivoadministration of the resulting liposomes such that a filter of about0.22 μm is employed;

a microemulsification method whereby an aqueous suspension of gaseousprecursor-filled liposomes of the present invention are emulsified byagitation and heated to form microspheres prior to administration to apatient; and

forming a gaseous precursor in lipid suspension by heating, and/oragitation, whereby the less dense gaseous precursor-filled microspheresfloat to the top of the solution by expanding and displacing othermicrospheres in the vessel and venting the vessel to release air.

Freeze drying is useful to remove water and organic materials from thelipids prior to the shaking gas instillation method. Drying-gasinstillation method may be used to remove water from liposomes. Bypre-entrapping the gaseous precursor in the dried liposomes (i.e. priorto drying) after warming, the gaseous precursor may expand to fill theliposome. Gaseous precursors can also be used to fill dried liposomesafter they have been subjected to vacuum. As the dried liposomes arekept at a temperature below their gel state to liquid crystallinetemperature the drying chamber can be slowly filled with the gaseousprecursor in its gaseous state, e.g. perfluorobutane can be used to filldried liposomes composed of dipalmitoylphosphatidylcholine (DPPC) attemperatures between 3° C. (the boiling point of perfluorobutane) andbelow 40° C., the phase transition temperature of the lipid. In thiscase, it would be most preferred to fill the liposomes at a temperatureabout 4° C. to about 5° C.

Preferred methods for preparing the temperature activated gaseousprecursor-filled liposomes comprise shaking an aqueous solution having alipid in the presence of a gaseous precursor at a temperature below thegel state to liquid crystalline state phase transition temperature ofthe lipid. The present invention also provides a method for preparinggaseous precursor-filled liposomes comprising shaking an aqueoussolution comprising a lipid in the presence of a gaseous precursor, andseparating the resulting gaseous precursor-filled liposomes fordiagnostic or therapeutic use. Liposomes prepared by the foregoingmethods are referred to herein as gaseous precursor-filled liposomesprepared by a gel state shaking gaseous precursor installation method.

Conventional, aqueous-filled liposomes are routinely formed at atemperature above the phase transition temperature of the lipid, sincethey are more flexible and thus useful in biological systems in theliquid crystalline state. See, for example, Szoka and Papahadjopoulos,Proc. Natl. Acad. Sci. 1978, 75, 4194-4198. In contrast, the liposomesmade according to preferred embodiments of the methods of the presentinvention are gaseous precursor-filled, which imparts greaterflexibility since gaseous precursors after gas formation are morecompressible and compliant than an aqueous solution. Thus, the gaseousprecursor-filled liposomes may be utilized in biological systems whenformed at a temperature below the phase transition temperature of thelipid, even though the gel phase is more rigid.

The methods of the present invention provide for shaking an aqueoussolution comprising a lipid in the presence of a temperature activatedgaseous precursor. Shaking, as used herein, is defined as a motion thatagitates an aqueous solution such that gaseous precursor is introducedfrom the local ambient environment into the aqueous solution. Any typeof motion that agitates the aqueous solution and results in theintroduction of gaseous precursor may be used for the shaking. Theshaking must be of sufficient force to allow the formation of foam aftera period of time. Preferably, the shaking is of sufficient force suchthat foam is formed within a short period of time, such as 30 minutes,and preferably within 20 minutes, and more preferably, within 10minutes. The shaking may be by microemulsifying, by microfluidizing, forexample, swirling (such as by vortexing), side-to-side, or up and downmotion. In the case of the addition of gaseous precursor in the liquidstate, sonication may be used in addition to the shaking methods setforth above. Further, different types of motion may be combined. Also,the shaking may occur by shaking the container holding the aqueous lipidsolution, or by shaking the aqueous solution within the containerwithout shaking the container itself. Further, the shaking may occurmanually or by machine. Mechanical shakers that may be used include, forexample, a shaker table, such as a VWR Scientific (Cerritos, Calif.)shaker table, a microfluidizer, Wig-L-Bug™ (Crescent DentalManufacturing, Inc., Lyons, Ill.) and a mechanical paint mixer, as wellas other known machines. Another means for producing shaking includesthe action of gaseous precursor emitted under high velocity or pressure.It will also be understood that preferably, with a larger volume ofaqueous solution, the total amount of force will be correspondinglyincreased. Vigorous shaking is defined as at least about 60 shakingmotions per minute, and is preferred. Vortexing at at least 1000revolutions per minute, an example of vigorous shaking, is morepreferred. Vortexing at 1800 revolutions per minute is most preferred.

The formation of gaseous precursor-filled liposomes upon shaking can bedetected by the presence of a foam on the top of the aqueous solution.This is coupled with a decrease in the volume of the aqueous solutionupon the formation of foam. Preferably, the final volume of the foam isat least about two times the initial volume of the aqueous lipidsolution; more preferably, the final volume of the foam is at leastabout three times the initial volume of the aqueous solution; even morepreferably, the final volume of the foam is at least about four timesthe initial volume of the aqueous solution; and most preferably, all ofthe aqueous lipid solution is converted to foam.

The required duration of shaking time may be determined by detection ofthe formation of foam. For example, 10 ml of lipid solution in a 50 mlcentrifuge tube may be vortexed for approximately 15-20 minutes or untilthe viscosity of the gaseous precursor-filled liposomes becomessufficiently thick so that it no longer clings to the side walls as itis swirled. At this time, the foam may cause the solution containing thegaseous precursor-filled liposomes to raise to a level of 30 to 35 ml.

The concentration of lipid required to form a preferred foam level willvary depending upon the type of lipid used, and may be readilydetermined by one skilled in the art, once armed with the presentdisclosure. For example, in preferred embodiments, the concentration of1,2-dipalimitoylphosphatidylcholine (DPPC) used to form gaseousprecursor-filled liposomes according to the methods of the presentinvention is about 20 mg/ml to about 30 mg/ml saline solution. Theconcentration of distearoylphosphatidylcholine (DSPC) used in preferredembodiments is about 5 mg/ml to about 10 mg/ml saline solution.

Specifically, DPPC in a concentration of 20 mg/ml to 30 mg/ml, uponshaking, yields a total suspension and entrapped gaseous precursorvolume four times greater than the suspension volume alone. DSPC in aconcentration of 10 mg/ml, upon shaking, yields a total volumecompletely devoid of any liquid suspension volume and contains entirelyfoam.

It will be understood by one skilled in the art, once armed with thepresent disclosure, that the lipids or liposomes may be manipulatedprior and subsequent to being subjected to the methods of the presentinvention. For example, the lipid may be hydrated and then lyophilized,processed through freeze and thaw cycles, or simply hydrated. Inpreferred embodiments, the lipid is hydrated and then lyophilized, orhydrated, then processed through freeze and thaw cycles and thenlyophilized, prior to the formation of gaseous precursor-filledliposomes.

According to the methods of the present invention, the presence of gas,such as and not limited to air, may also be provided by the localambient atmosphere. The local ambient atmosphere may be the atmospherewithin a sealed container, or in an unsealed container, may be theexternal environment. Alternatively, for example, a gas may be injectedinto or otherwise added to the container having the aqueous lipidsolution or into the aqueous lipid solution itself in order to provide agas other than air. Gases that are not heavier than air may be added toa sealed container while gases heavier than air may be added to a sealedor an unsealed container. Accordingly, the present invention includesco-entrapment of air and/or other gases along with gaseous precursors.

The preferred methods of the invention are carried out at a temperaturebelow the gel state to liquid crystalline state phase transitiontemperature of the lipid employed. By "gel state to liquid crystallinestate phase transition temperature", it is meant the temperature atwhich a lipid bilayer will convert from a gel state to a liquidcrystalline state. See, for example, Chapman et al., J. Biol. Chem.1974, 249, 2512-2521. The gel state to liquid crystalline state phasetransition temperatures of various lipids will be readily apparent tothose skilled in the art and are described, for example, in Gregoriadis,ed., Liposome Technology, Vol. I, 1-18 (CRC Press, 1984) and DerekMarsh, CRC Handbook of Lipid Bilayers (CRC Press, Boca Raton, Fla.1990), at p. 139. See also Table I, below. Where the gel state to liquidcrystalline state phase transition temperature of the lipid employed ishigher than room temperature, the temperature of the container may beregulated, for example, by providing a cooling mechanism to cool thecontainer holding the lipid solution.

Since gaseous precursors (e.g. perfluorobutane) are less soluble anddiffusable than other gases, such as air, they tend to be more stablewhen entrapped in liposomes even when the liposomes are composed oflipids in the liquid-crystalline state. Small liposomes composed ofliquid-crystalline state lipid such as egg phosphatidyl choline may beused to entrap a nanodroplet of perfluorobutane. For example, lipidvesicles with diameters of about 30 nm to about 50 nm may be used toentrap nanodroplets of perfluorobutane with with mean diameter of about25 nm. After temperature activated conversion, the precursor filledliposomes will create microspheres of about 10 microns in diameter. Thelipid in this case, serves the purpose of defining the size of themicrosphere via the small liposome. The lipids also serve to stabilizethe resultant microsphere size. In this case, techniques such asmicroemulsification are preferred for forming the small liposomes whichentrap the precursor. A microfluidizer (Microfluidics, Newton, Mass.) isparticularly useful for making an emulsion of small liposomes whichentrap the gaseous precursor.

                  TABLE I                                                         ______________________________________                                        Saturated Diacyl-sn-Glycero-3-Phosphocholines                                 Main Chain Gel State to Liquid Crystalline State                              Phase Transition Temperatures                                                                Liquid Crystalline                                             # Carbons in Acyl                                                                            Phase Transition                                               Chains         Temperature (°C.)                                       ______________________________________                                        1,2-(12:0)     -1.0                                                           1,2-(13:0)     13.7                                                           1,2-(14:0)     23.5                                                           1,2-(15:0)     34.5                                                           1,2-(16:0)     41.4                                                           1,2-(17:0)     48.2                                                           1,2-(18:0)     55.1                                                           1,2-(19:0)     61.8                                                           1,2-(20:0)     64.5                                                           1,2-(21:0)     71.1                                                           1,2-(22:0)     74.0                                                           1,2-(23:0)     79.5                                                           1,2-(24:0)     80.1                                                           ______________________________________                                    

Conventional, aqueous-filled liposomes are routinely formed at atemperature above the gel to liquid crystalline phase transitiontemperature of the lipid, since they are more flexible and thus usefulin biological systems in the liquid crystalline state. See, for example,Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. 1978, 75, 4194-4198.In contrast, the liposomes made according to preferred embodiments ofthe methods of the present invention are gaseous precursor-filled, whichimparts greater flexibility since gaseous precursor is more compressibleand compliant than an aqueous solution. Thus, the gaseousprecursor-filled liposomes may be utilized in biological systems whenformed at a temperature below the phase transition temperature of thelipid, even though the gel phase is more rigid.

A preferred apparatus for producing the temperature activated gaseousprecursor-filled liposomes using a gel state shaking gaseous precursorinstillation process is shown in FIG. 1. A mixture of lipid and aqueousmedia is vigorously agitated in the presence of gaseous precursor toproduce gaseous precursor-filled liposomes, either by batch or bycontinuous feed. Referring to FIG. 1, dried lipids 51 from a lipidsupply vessel 50 are added via conduit 59 to a mixing vessel 66 ineither a continuous flow or as intermittent boluses. If a batch processis utilized, the mixing vessel 66 may comprise a relatively smallcontainer such as a syringe, test tube, bottle or round bottom flask, ora large container. If a continuous feed process is utilized, the mixingvessel is preferably a large container, such as a vat.

Where the gaseous precursor-filled liposomes contain a therapeuticcompound, the therapeutic compound may be added, for example, in amanner similar to the addition of the lipid described above before thegaseous precursor installation process. Alternatively, the therapeuticcompound may be added after the gaseous precursor installation processwhen the liposomes are coated on the outside with the therapeuticcompound.

In addition to the lipids 51, an aqueous media 53, such as a salinesolution, from an aqueous media supply vessel 52, is also added to thevessel 66 via conduit 61. The lipids 51 and the aqueous media 53 combineto form an aqueous lipid solution 74. Alternatively, the dried lipids 51could be hydrated prior to being introduced into the mixing vessel 66 sothat lipids are introduced in an aqueous solution. In the preferredembodiment of the method for making liposomes, the initial charge ofsolution 74 is such that the solution occupies only a portion of thecapacity of the mixing vessel 66. Moreover, in a continuous process, therates at which the aqueous lipid solution 74 is added and gaseousprecursor-filled liposomes produced are removed is controlled to ensurethat the volume of lipid solution 74 does not exceed a predeterminedpercentage of the mixing vessel 66 capacity.

The shaking may be accomplished by introducing a high velocity jet of apressurized gaseous precursor directly into the aqueous lipid solution74. Alternatively, the shaking may be accomplished by mechanicallyshaking the aqueous solution, either manually or by machine. Suchmechanical shaking may be effected by shaking the mixing vessel 66 or byshaking the aqueous solution 74 directly without shaking the mixingvessel itself. As shown in FIG. 1, in the preferred embodiment, amechanical shaker 75, is connected to the mixing vessel 66. The shakingshould be of sufficient intensity so that, after a period of time, afoam 73 comprised of gaseous precursor-filled liposomes is formed on thetop of the aqueous solution 74, as shown in FIG. 1. The detection of theformation of the foam 73 may be used as a means for controlling theduration of the shaking; that is, rather than shaking for apredetermined period of time, the shaking may be continued until apredetermined volume of foam has been produced.

The apparatus of FIG. 1 may also contain a means for controllingtemperature such that the apparatus may be maintained at one temperaturefor the method of making the liposomes. For example, in the preferredembodiment, the methods of making liposomes are performed at atemperature below the boiling point of the gaseous precursor. In thepreferred embodiment, a liquid gaseous precursor fills the internalspace of the liposomes. Alternatively, the apparatus may be maintainedat about the temperature of the liquid to gas transition temperature ofthe gaseous precursor such that a gas is contained in the liposomes.Further, the temperature of the apparatus may be adjusted throughout themethod of making the liposomes such that the gaseous precursor begins asa liquid, however, a gas is incorporated into the resulting liposomes.In this embodiment, the temperature of the apparatus is adjusted duringthe method of making the liposomes such that the method begins at atemperature below the phase transition temperature and is adjusted to atemperature at about the phase transition temperature of the gaseousprecursor. Accordingly, the vessel may be closed and periodicallyvented, or open to the ambient atmosphere.

In a preferred embodiment of the apparatus for making gaseousprecursor-filled liposomes in which the lipid employed has a gel toliquid crystalline phase transition temperature below room temperature,a means for cooling the aqueous lipid solution 74 is provided. In theembodiment shown in FIG. 1, cooling is accomplished by means of a jacket64 disposed around the mixing vessel 66 so as to form an annular passagesurrounding the vessel. As shown in FIG. 1, a cooling fluid 63 is forcedto flow through this annular passage by means of jacket inlet and outletports 62 and 63, respectively. By regulating the temperature and flowrate of the cooling fluid 62, the temperature of the aqueous lipidsolution 74 can be maintained at the desired temperature.

As shown in FIG. 1, a gaseous precursor 55, which may be 1-fluorobutane,for example, is introduced into the mixing vessel 66 along with theaqueous solution 74. Air may be introduced by utilizing an unsealedmixing vessel so that the aqueous solution is continuously exposed toenvironmental air. In a batch process, a fixed charge of local ambientair may be introduced by sealing the mixing vessel 66. If a gaseousprecursor heavier than air is used, the container need not be sealed.However, introduction of gaseous precursors that are not heavier thanair will require that the mixing vessel be sealed, for example by use ofa lid 65, as shown in FIG. 1. The gaseous precursor 55 may bepressurized in the mixing vessel 66, for example, by connecting themixing vessel to a pressurized gas supply tank 54 via a conduit 57, asshown in FIG. 1.

After the shaking is completed, the gaseous precursor-filled liposomecontaining foam 73 may be extracted from the mixing vessel 66.Extraction may be accomplished by inserting the needle 102 of a syringe100, shown in FIG. 2, into the foam 73 and drawing a predeterminedamount of foam into the barrel 104 by withdrawing the plunger 106. Asdiscussed further below, the location at which the end of the needle 102is placed in the foam 73 may be used to control the size of the gaseousprecursor-filled liposomes extracted.

Alternatively, extraction may be accomplished by inserting an extractiontube 67 into the mixing vessel 66, as shown in FIG. 1. If the mixingvessel 66 is pressurized, as previously discussed, the pressure of thegaseous precursor 55 may be used to force the gaseous precursor-filledliposomes 77 from the mixing vessel 66 to an extraction vessel 76 viaconduit 70. In the event that the mixing vessel 66 is not pressurized,the extraction vessel 76 may be connected to a vacuum source 58, such asa vacuum pump, via conduit 78, that creates sufficient negative pressureto suck the foam 73 into the extraction vessel 76, as shown in FIG. 1.From the extraction vessel 76, the gaseous precursor-filled liposomes 77are introduced into vials 82 in which they may be shipped to theultimate user. A source of pressurized gaseous precursor 56 may beconnected to the extraction vessel 76 as aid to ejecting the gaseousprecursor-filled liposomes. Since negative pressure may result inincreasing the size of the gaseous precursor-filled liposomes, positivepressure is preferred for removing the gaseous precursor-filledliposomes.

Filtration may be carried out in order to obtain gaseousprecursor-filled liposomes of a substantially uniform size. In certainpreferred embodiments, the filtration assembly contains more than onefilter, and preferably, the filters are not immediately adjacent to eachother, as illustrated in FIG. 4. Before filtration, the gaseousprecursor-filled liposomes range in size from about 1 micron to greaterthan 60 microns (FIGS. 5A and 6A). After filtration through a singlefilter, the gaseous precursor-filled liposomes are generally less than10 microns but particles as large as 25 microns in size remain. Afterfiltration through two filters (10 micron followed by 8 micron filter),almost all of the liposomes are less than 10 microns, and most are 5 to7 microns (FIGS. 5B and 6B).

As shown in FIG. 1, filtering may be accomplished by incorporating afilter element 72 directly onto the end of the extraction tube 67 sothat only gaseous precursor-filled liposomes below a pre-determined sizeare extracted from the mixing vessel 66. Alternatively, or in additionto the extraction tube filter 72, gaseous precursor-filled liposomesizing may be accomplished by means of a filter 80 incorporated into theconduit 79 that directs the gaseous precursor-filled liposomes 77 fromthe extraction vessel 76 to the vials 82, as shown in FIG. 1. The filter80 may contain a cascade filter assembly 124, such as that shown in FIG.4. The cascade filter assembly 124 shown in FIG. 4 comprises twosuccessive filters 116 and 120, with filter 120 being disposed upstreamof filter 116. In a preferred embodiment, the upstream filter 120 is a"NUCLEPORE" 10 μm filter and the downstream filter 116 is a "NUCLEPORE"8 μm filter. Two 0.15 mm metallic mesh discs 115 are preferablyinstalled on either side of the filter 116. In a preferred embodiment,the filters 116 and 120 are spaced apart a minimum of 150 μm by means ofa Teflon™ O-ring, 118.

In addition to filtering, sizing may also be accomplished by takingadvantage of the dependence of gaseous precursor-filled liposomebuoyancy on size. The gaseous precursor-filled liposomes haveappreciably lower density than water and hence may float to the top ofthe mixing vessel 66. Since the largest liposomes have the lowestdensity, they will float most quickly to the top. The smallest liposomeswill generally be last to rise to the top and the non gaseousprecursor-filled lipid portion will sink to the bottom. This phenomenonmay be advantageously used to size the gaseous precursor-filledliposomes by removing them from the mixing vessel 66 via a differentialflotation process. Thus, the setting of the vertical location of theextraction tube 67 within the mixing vessel 66 may control the size ofthe gaseous precursor-filled liposomes extracted; the higher the tube,the larger the gaseous precursor-filled liposomes extracted. Moreover,by periodically or continuously adjusting the vertical location of theextraction tube 67 within the mixing vessel 66, the size of the gaseousprecursor-filled liposomes extracted may be controlled on an on-goingbasis. Such extraction may be facilitated by incorporating a device 68,which may be a threaded collar 71 mating with a threaded sleeve 72attached to the extraction tube 67, that allows the vertical location ofthe extraction tube 66 within the extraction vessel 66 to be accuratelyadjusted.

The gel state shaking gaseous precursor installation process itself mayalso be used to improve sizing of the gaseous precursor-filled lipidbased microspheres. In general, the greater the intensity of the shakingenergy, the smaller the size of the resulting gaseous precursor-filledliposomes.

The current invention also includes novel methods for preparingdrug-containing gaseous precursor-filled liposomes to be dispensed tothe ultimate user. Once gaseous precursor-filled liposomes are formed,they generally cannot be sterilized by heating at a temperature thatwould cause rupture. Therefore, it is desirable to form the gaseousprecursor-filled liposomes from sterile ingredients and to perform aslittle subsequent manipulation as possible to avoid the danger ofcontamination. According to the current invention, this may beaccomplished, for example, by sterilizing the mixing vessel containingthe lipid and aqueous solution before shaking and dispensing the gaseousprecursor-filled liposomes 77 from the mixing vessel 66, via theextraction vessel 76, directly into the barrel 104 of a sterile syringe100, shown in FIG. 2, without further processing or handling; that is,without subsequent sterilization. The syringe 100, charged with gaseousprecursor-filled liposomes 77 and suitably packaged, may then bedispensed to the ultimate user. Thereafter, no further manipulation ofthe product is required in order to administer the gaseousprecursor-filled liposomes to the patient, other than removing thesyringe from its packaging and removing a protector (not shown) from thesyringe needle 102 and inserting the needle into the body of thepatient, or into a catheter. Moreover, the pressure generated when thesyringe plunger 106 is pressed into the barrel 104 will cause thelargest gaseous precursor-filled liposomes to collapse, therebyachieving a degree of sizing without filtration.

Where it is desired to filter the gaseous precursor-filled liposomes atthe point of use, for example because they are removed from theextraction vessel 76 without filtration or because further filtration isdesired, the syringe 100 may be fitted with its own filter 108, as shownin FIG. 2. This results in the gaseous precursor-filled liposomes beingsized by causing them to be extruded through the filter 108 by theaction of the plunger 106 when the gaseous precursor-filled liposomesare injected. Thus, the gaseous precursor-filled liposomes may be sizedand injected into a patient in one step.

In order to accommodate the use of a single or dual filter in the hubhousing of the syringe, a non-standard syringe with hub housing isnecessary. As shown in FIG. 3, the hub that houses the filter(s) are ofa dimension of approximately 1 cm to approximately 2 cm in diameter byabout 1.0 cm to about 3.0 cm in length with an inside diameter of about0.8 cm for which to house the filters. The abnormally large dimensionsfor the filter housing in the hub are to accommodate passage of themicrospheres through a hub with sufficient surface area so as todecrease the pressure that need be applied to the plunger of thesyringe. In this manner, the microspheres will not be subjected to aninordinately large pressure head upon injection, which may cause ruptureof the microspheres.

As shown in FIG. 3, a cascade filter housing 110 may be fitted directlyonto a syringe 112, thereby allowing cascade filtration at the point ofuse. As shown in FIG. 4, the filter housing 110 is comprised of acascade filter assembly 124, previously discussed, incorporated betweena lower collar 122, having male threads, and a female collar 114, havingfemale threads. The lower collar 122 is fitted with a Luer lock thatallows it to be readily secured to the syringe 112 and the upper collar114 is fitted with a needle 102.

In preferred embodiments, the lipid solution is extruded through afilter and the lipid solution is heat sterilized prior to shaking. Oncegaseous precursor-filled liposomes are formed, they may be filtered forsizing as described above. These steps prior to the formation of gaseousprecursor-filled liposomes provide the advantages, for example, ofreducing the amount of unhydrated lipid and thus providing asignificantly higher yield of gaseous precursor-filled liposomes, aswell as and providing sterile gaseous precursor-filled liposomes readyfor administration to a patient. For example, a mixing vessel such as avial or syringe may be filled with a filtered lipid suspension, and thesolution may then be sterilized within the mixing vessel, for example,by autoclaving. A gaseous precursor may be instilled into the lipidsuspension to form gaseous precursor-filled liposomes by shaking thesterile vessel. Preferably, the sterile vessel is equipped with a filterpositioned such that the gaseous precursor-filled liposomes pass throughthe filter before contacting a patient.

The first step of this preferred method, extruding the lipid solutionthrough a filter, decreases the amount of unhydrated lipid by breakingup the dried lipid and exposing a greater surface area for hydration.Preferably, the filter has a pore size of about 0.1 to about 5 μm, morepreferably, about 0.1 to about 4 μm, even more preferably, about 0.1 toabout 2 μm, and even more preferably, about 1 μm, most preferably 0.22μm. As shown in FIG. 7, when a lipid suspension is filtered (FIG. 7B),the amount of unhydrated lipid is reduced when compared to a lipidsuspension that was not pre-filtered (FIG. 7A). Unhydrated lipid appearsas amorphous clumps of non-uniform size and is undesirable.

The second step, sterilization, provides a composition that may bereadily administered to a patient. Preferably, sterilization isaccomplished by heat sterilization, preferably, by autoclaving thesolution at a temperature of at least about 100° C., and morepreferably, by autoclaving at about 100° C. to about 130° C., even morepreferably, about 110° C. to about 130° C., even more preferably, about120° C. to about 130° C., and most preferably, about 130° C. Preferably,heating occurs for at least about 1 minute, more preferably, about 1 toabout 30 minutes, even more preferably, about 10 to about 20 minutes,and most preferably, about 15 minutes.

Where sterilization occurs by a process other than heat sterilization ata temperature which would cause rupture of the gaseous precursor-filledliposomes, sterilization may occur subsequent to the formation of thegaseous precursor-filled liposomes, and is preferred. For example, gammaradiation may be used before and/or after gaseous precursor-filledliposomes are formed.

Sterilization of the gaseous precursor may be achieved via passagethrough a 0.22 μm filter or a smaller filter, prior to emulsification inthe aqueous media. This can be easily achieved via sterile filtration ofthe contents directly into a vial which contains a predetermined amountof likewise sterilized and sterile-filled aqueous carrier.

FIG. 8 illustrates the ability of gaseous precursor-filled liposomes tosuccessfully form after autoclaving, which was carried out at 130° C.for 15 minutes, followed by vortexing for 10 minutes. Further, after theextrusion and sterilization procedure, the shaking step yields gaseousprecursor-filled liposomes with little to no residual anhydrous lipidphase. FIG. 8A shows gaseous precursor-filled liposomes generated afterautoclaving but prior to filtration, thus resulting in a number ofgaseous precursor-filled liposomes having a size greater than 10 μm.FIG. 8B shows gaseous precursor-filled liposomes after a filtrationthrough a 10 μm "NUCLEPORE" filter, resulting in a uniform size around10 μm.

The materials which may be utilized in preparing the gaseousprecursor-filled lipid microspheres include any of the materials orcombinations thereof known to those skilled in the art as suitable forliposome preparation. Gas precursors which undergo phase transition froma liquid to a gas at their boiling point may be used in the presentinvention. The lipids used may be of either natural or synthetic origin.The particular lipids are chosen to optimize the desired properties,e.g., short plasma half-life versus long plasma half-life for maximalserum stability. It will also be understood that certain lipids may bemore efficacious for particular applications, such as the containment ofa therapeutic compound to be released upon rupture of the gaseousprecursor-filled lipid microsphere.

The lipid in the gaseous precursor-filled liposomes may be in the formof a single bilayer or a multilamellar bilayer, and are preferablymultilamellar.

Gaseous precursors which may be activated by temperature may be usefulin the present invention. Table II lists examples of gaseous precursorswhich undergo phase transitions from liquid to gaseous states at closeto normal body temperature (37° C.) and the size of the emulsifieddroplets that would be required to form a microsphere having a size of10 microns. The list is composed of potential gaseous precursors thatmay be used to form temperature activated gaseous precursor-containingliposomes of a defined size. The list should not be construed as beinglimiting by any means, as to the possibilities of gaseous precursors forthe methods of the present invention.

                  TABLE II                                                        ______________________________________                                        Physical Characteristics of Gaseous Precursors and                            Diameter of Emulsified Droplet to Form a 10 μm Microsphere                                                  Diameter (μm) of                                    Molec-  Boiling        Emulsified droplet                                     ular    Point          to make 10 micron                            Compound  Weight  (°C.)                                                                          Density                                                                              microsphere                                  ______________________________________                                        1-        76.11   32.5    6.7789 1.2                                          fluorobutane                                                                  2-methyl  72.15   27.8    0.6201 2.6                                          butane                                                                        (isopentane)                                                                  2-methyl 1-                                                                             70.13   31.2    0.6504 2.5                                          butene                                                                        2-methyl-2-                                                                             70.13   38.6    0.6623 2.5                                          butene                                                                        1-butene-3-                                                                             66.10   34.0    0.6801 2.4                                          yne-2-methyl                                                                  3-methyl-1-                                                                             68.12   29.5    0.6660 2.5                                          butyne                                                                        perfluoro 88.00   -129    3.034  3.3                                          methane                                                                       perfluoro 138.01  -79     1.590  1.0                                          ethane                                                                        perfluoro 238.03  3.96    1.6484 2.8                                          butane                                                                        perfluoro 288.04  57.73   1.7326 2.9                                          pentane                                                                       octafluoro                                                                              200.04  -5.8    1.48   2.8                                          cyclobutane                                                                   decafluoro                                                                              238.04  2       1.517  3.0                                          butane                                                                        hexafluoro                                                                              138.01  -78.1   1.607  2.7                                          ethane                                                                        docecafluoro                                                                            288.05  29.5    1.664  2.9                                          pentane                                                                       octafluoro-2-                                                                           200.04  1.2     1.5297 2.8                                          butene                                                                        perfluoro 200.04  -5.8    1.48   2.8                                          cyclobutane                                                                   octafluoro                                                                              212.05  27      1.58   2.7                                          cyclopentene                                                                  perfluoro 162     5       1.602  2.5                                          cyclobutene                                                                   ______________________________________                                         *Source: Chemical Rubber Company Handbook of Chemistry and Physics Robert     C. Weast and David R. Lide, eds. CRC Press, Inc. Boca Raton, Florida.         (1989-1990).                                                             

Examples of gaseous precursors are by no means limited to Table II. Infact, for a variety of different applications, virtually any liquid canbe used to make gaseous precursors so long as it is capable ofundergoing a phase transition to the gas phase upon passing through theappropriate activation temperature. Examples of gaseous precursors thatmay be used include, and are by no means limited to, the following:hexafluoro acetone; isopropyl acetylene; allene; tetrafluoroallene;boron trifluoride; 1,2-butadiene; 1,3-butadiene; 1,3-butadiene;1,2,3-trichloro, 2-fluoro-1,3-butadiene; 2-methyl,1,3 butadiene;hexafluoro-1,3-butadiene; butadiyne; 1-fluoro-butane; 2-methyl-butane;decafluoro butane; 1-butene; 2-butene; 2-methy-1-butene;3-methyl-1-butene; perfluoro-1-butene; perfluoro-1-butene;perfluoro-2-butene; 1,4-phenyl-3-butene-2-one; 2-methyl-1-butene-3-yne;butyl nitrate; 1-butyne; 2-butyne;2-chloro-1,1,1,4,4,4-hexafluoro-butyne; 3-methyl-1-butyne;perfluoro-2-butyne; 2-bromo-butyraldehyde; carbonyl sulfide;crotononitrile; cyclobutane; methyl-cyclobutane; octafluoro-cyclobutane;perfluoro-cyclobutene; 3-chloro-cyclopentene; perfluoro ethane;perfluoro propane; perfluoro butane; perfluoro pentane; perfluorohexane; cyclopropane; 1,2-dimethyl-cyclopropane; 1,1-dimethylcyclopropane; 1,2-dimethyl cyclopropane; ethyl cyclopropane; methylcyclopropane; diacetylene; 3-ethyl-3-methyl diaziridine;1,1,1-trifluorodiazoethane; dimethyl amine; hexafluoro-dimethyl amine;dimethylethylamine; -bis-(Dimethyl phosphine)amine;2,3-dimethyl-2-norbornane; perfluorodimethylamine; dimethyloxoniumchloride; 1,3-dioxolane-2-one; perfluorocarbons such as and not limitedto 4-methyl,1,1,1,2-tetrafluoro ethane; 1,1,1-trifluoroethane;1,1,2,2-tetrafluoroethane; 1,1,2-trichloro-1,2,2-trifluoroethane; 1,1dichloroethane; 1,1-dichloro-1,2,2,2-tetrafluoro ethane; 1,2-difluoroethane; 1-chloro-1,1,2,2,2-pentafluoro ethane; 2-chloro,1,1-difluoroethane; 1-chloro-1,1,2,2-tetrafluoro ethane; 2-chloro,1,1-difluoroethane; chloroethane; chloropentafluoro ethane;dichlorotrifluoroethane; fluoro-ethane; hexafluoro-ethane;nitro-pentafluoro ethane; nitroso-pentafluoro ethane; perfluoro ethane;perfluoro ethylamine; ethyl vinyl ether; 1,1-dichloro ethylene;1,1-dichloro-1,2-difluoro ethylene; 1,2-difluoro ethylene; Methane;Methane-sulfonyl chloride-trifluoro; Methanesulfonyl fluoride-trifluoro;Methane-(pentafluorothio)trifluoro; Methane-bromo difluoro nitroso;Methane-bromo fluoro; Methane-bromo chloro-fluoro;Methanebromo-trifluoro; Methane-chloro difluoro nitro; Methane-chlorodinitro; Methanechloro fluoro; Methane-chloro trifluoro;Methane-chloro-difluoro; Methane dibromo difluoro; Methane-dichlorodifluoro; Methane-dichloro-fluoro; Methanedifluoro;Methane-difluoro-iodo; Methane-disilano; Methane-fluoro; Methaneiodo;Methane-iodo-trifluoro; Methane-nitro-trifluoro;Methane-nitroso-trifluoro; Methane-tetrafluoro; Methane-trichlorofluoro;Methane-trifluoro; Methanesulfenylchloride-trifluoro; 2-Methyl butane;Methyl ether; Methyl isopropyl ether; Methyl lactate; Methyl nitrite;Methyl sulfide; Methyl vinyl ether; Neon; Neopentane; Nitrogen (N₂);Nitrous oxide; 1,2,3-Nonadecane tricarboxylicacid-2-hydroxytrimethylester; 1-Nonene-3-yne; Oxygen (O₂);1,4-Pentadiene; n-Pentane; Pentane-perfluoro;2-Pentanone-4-amino-4-methyl; 1-Pentene; 2-Pentene [cis]; 2-Pentene(trans); 1-Pentene-3-bromo; 1-Pentene-perfluoro; Phthalicacid-tetrachloro; Piperidine-2,3,6-trimethyl; Propane,Propane-1,1,1,2,2,3-hexafluoro; Propane-1,2-epoxy; Propane-2,2 difluoro;Propane 2-amino, Propane-2-chloro; Propane-heptafluoro-1-nitro;Propane-heptafluoro-1-nitroso; Propane-perfluoro; Propene;Propyl-1,1,1,2,3,3-hexafluoro-2,3 dichloro; Propylene-1-chloro;Propylenechloro-(trans); Propylene-2-chloro; Propylene-3-fluoro;Propylene-perfluoro; Propyne; Propyne-3,3,3-trifluoro; Styrene-3-fluoro;Sulfur hexafluoride; Sulfur (di)-decafluoro(S2F10); Toluene-2,4-diamino;Trifluoroacetonitrile; Trifluoromethyl peroxide; Trifluoromethylsulfide; Tungsten hexafluoride; Vinyl acetylene; Vinyl ether; Xenon;Nitrogen; air; and other ambient gases.

Perfluorocarbons are the preferred gases of the present invention,fluorine gas, perfluoromethane, perfluoroethane, perfluorobutane,perfluoropentane, perfluorohexane; even more preferrablyperfluoroethane, perfluoropropane and perfluorobutane; most preferrablyperfluoropropane and perfluorobutane as the more inert perfluorinatedgases are less toxic.

Microspheres of the present invention include and are not limited toliposomes, lipid coatings, emulsions and polymers.

Lipids which may be used to create lipid microspheres include but arenot limited to: lipids such as fatty acids, lysolipids,phosphatidylcholine with both saturated and unsaturated lipids includingdioleoylphosphatidylcholine; dimyristoylphosphatidylcholine;dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine;distearoylphosphatidylcholine; phosphatidylethanolamines such asdioleoylphosphatidylethanolamine; phosphatidylserine;phosphatidylglycerol; phosphatidylinositol, sphingolipids such assphingomyelin; glycolipids such as ganglioside GM1 and GM2; glucolipids;sulfatides; glycosphingolipids; phosphatidic acid; palmitic acid;stearic acid; arachidonic acid; oleic acid; lipids bearing polymers suchas polyethyleneglycol, chitin, hyaluronic acid or polyvinylpyrrolidone;lipids bearing sulfonated mono-, di-, oligo- or polysaccharides;cholesterol, cholesterol sulfate and cholesterol hemisuccinate;tocopherol hemisuccinate, lipids with ether and ester-linked fattyacids, polymerized lipids, diacetyl phosphate, stearylamine,cardiolipin, phospholipids with short chain fatty acids of 6-8 carbonsin length, synthetic phospholipids with asymmetric acyl chains (e.g.,with one acyl chain of 6 carbons and another acyl chain of 12 carbons),6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside,digalactosyldiglyceride,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside,12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoicacid; N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoyl]-2-aminopalmitic acid;cholesteryl)4'-trimethyl-ammonio)butanoate;N-succinyldioleoylphosphatidylethanol-amine;1,2-dioleoyl-sn-glycerol;1,2-dipalmitoyl-sn-3-succinylglycerol;1,3-dipalmitoyl-2-succinylglycerol;1-hexadecyl-2-palmitoylglycerophosphoethanolamine;and palmitoylhomocysteine; and/or combinations thereof. The liposomesmay be formed as monolayers or bilayers and may or may not have acoating.

Lipids bearing hydrophilic polymers such as polyethyleneglycol (PEG),including and not limited to PEG 2,000 MW, 5,000 MW, and PEG 8,000 MW,are particularly useful for improving the stability and sizedistribution of the gaseous precursor-containing liposomes. Variousdifferent mole ratios of PEGylated lipid,dipalmitoylphosphatidylethanolamine (DPPE) bearing PEG 5,000 MW, forexample, are also useful; 8 mole percent DPPE is preferred. A preferredproduct which is highly useful for entrapping gaseous precursorscontains 83 mole percent DPPC, 8 mole percent DPPE-PEG 5,000 MW and 5mole percent dipalmitoylphosphatidic acid.

In addition, examples of compounds used to make mixed systems include,but by no means are limited to lauryltrimethylammonium bromide(dodecyl-), cetyltrimethylammonium bromide (hexadecyl-),myristyltrimethylammonium bromide (tetradecyl-),alkyldimethylbenzylammonium chloride (alkyl=C12,C14,C16),benzyldimethyldodecylammonium bromide/chloride,benzyldimethylhexadecylammonium bromide/chloride,benzyldimethyltetradecylammonium bromide/chloride,cetyldimethylethylammonium bromide/chloride, or cetylpyridiniumbromide/chloride. Likewise perfluorocarbons such as pentafluorooctadecyl iodide, perfluorooctylbromide (PFOB), perfluorodecalin,perfluorododecalin, perfluorooctyliodide, perfluorotripropylamine, andperfluorotributylamine. The perfluorocarbons may be entrapped inliposomes or stabilized in emulsions as is well know in the art such asU.S. Pat. No. 4,865,836. The above examples of lipid suspensions mayalso be sterilized via autoclave without appreciable change in the sizeof the suspensions.

If desired, either anionic or cationic lipids may be used to bindanionic or cationic pharmaceuticals. Cationic lipids may be used to bindDNA and RNA analogues with in or on the surface of the gaseousprecursor-filled microsphere. A variety of lipids such as DOTMA,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTAP,1,2-dioleoyloxy-3-(trimethylammonio)propane; and DOTB,1,2-dioleoyl-3-(4'-trimethyl-ammonio)butanoyl-sn-glycerol may be used.In general the molar ratio of cationic lipid to non-cationic lipid inthe liposome may be, for example, 1:1000, 1:100, preferably, between 2:1to 1:10, more preferably in the range between 1:1 to 1:2.5 and mostpreferably 1:1 (ratio of mole amount cationic lipid to mole amountnon-cationic lipid, e.g., DPPC). A wide variety of lipids may comprisethe non-cationic lipid when cationic lipid is used to construct themicrosphere. Preferably, this non-cationic lipid isdipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine ordioleoylphosphatidylethanolamine. In lieu of cationic lipids asdescribed above, lipids bearing cationic polymers such as polylysine orpolyarginine may also be used to construct the microspheres and affordbinding of a negatively charged therapeutic, such as genetic material,to the outside of the microspheres. Additionally, negatively chargedlipids may be used, for example, to bind positively charged therapeuticcompounds. Phosphatidic acid, a negatively charged lipid, can also beused to complex DNA. This is highly surprising, as the positivelycharged lipids were heretofore thought to be generally necessary to bindgenetic materials to liposomes. 5 to 10 mole percent phosphatidic acidin the liposomes improves the stability and size distribution of thegaseous precursor-filled liposomes.

Other useful lipids or combinations thereof apparent to those skilled inthe art which are in keeping with the spirit of the present inventionare also encompassed by the present invention. For example,carbohydrate-bearing lipids may be employed for in vivo targeting, asdescribed in U.S. Pat. No. 4,310,505, the disclosures of which arehereby incorporated herein by reference in their entirety.

The most preferred lipids are phospholipids, preferably DPPC and DSPC,and most preferably DPPC.

Saturated and unsaturated fatty acids that may be used to generategaseous precursor-filled microspheres preferably include, but are notlimited to molecules that have between 12 carbon atoms and 22 carbonatoms in either linear or branched form. Examples of saturated fattyacids that may be used include, but are not limited to, lauric,myristic, palmitic, and stearic acids. Examples of unsaturated fattyacids that may be used include, but are not limited to, lauroleic,physeteric, myristoleic, palmitoleic, petroselinic, and oleic acids.Examples of branched fatty acids that may be used include, but are notlimited to, isolauric, isomyristic, isopalmitic, and isostearic acidsand isoprenoids.

Cationic polymers may be bound to the lipid layer through one or morealkyl groups or sterol groups which serve to anchor the cationic polymerinto the lipid layer surrounding the gaseous precursor. Cationicpolymers that may be used in this manner include, but are not limitedto, polylysine and polyarginine, and their analogs such aspolyhomoarginine or polyhomolysine. The positively charged groups ofcationic lipids and cationic polymers, or perfluoroalkylated groupsbearing cationic groups, for example, may be used to complex negativelycharged molecules such as sugar phosphates on genetic material, thusbinding the material to the surface of the gaseous precursor-filledlipid sphere. For example, cationic analogs of amphiphilicperfluoroalkylated bipyridines, as described in Garelli and Vierling,Biochim. Biophys Acta, 1992, 1127, 41-48, the disclosures of which arehereby incorporated herein by reference in their entirety, may be used.Alternatively, for example, negatively charged molecules may be bounddirectly to the head groups of the lipids via ester, amide, ether,disulfide or thioester linkages.

Bioactive materials, such as peptides or proteins, may be incorporatedinto the lipid layer provided the peptides have sufficient lipophilicityor may be derivatized with alkyl or sterol groups for attachment to thelipid layer. Negatively charged peptides may be attached, for example,using cationic lipids or polymers as described above.

One or more emulsifying or stabilizing agents may be included with thegaseous precursors to formulate the temperature activated gaseousprecursor-filled microspheres. The purpose of theseemulsifying/stabilizing agents is two-fold. Firstly, these agents helpto maintain the size of the gaseous precursor-filled microsphere. Asnoted above, the size of these microspheres will generally affect thesize of the resultant gas-filled microspheres. Secondly the emulsifyingand stabilizing agents may be used to coat or stabilize the microspherewhich results from the precursor. Stabilization of contrastagent-containing microspheres is desirable to maximize the in vivocontrast effect. Although stabilization of the microsphere is preferredthis is not an absolute requirement. Because the gas-filled microspheresresulting from these gaseous precursors are more stable than air, theymay still be designed to provide useful contrast enhancement; forexample, they pass through the pulmonary circulation followingperipheral venous injection, even when not specifically stabilized byone or more coating or emulsifying agents. One or more coating orstabilizing agents is preferred however, as are flexible stabilizingmaterials. Gas microspheres stabilized by polysaccharides, gangliosides,and polymers are more effective than those stabilized by albumin andother proteins. Liposomes prepared using aliphatic compounds arepreferred as microspheres stabilized with these compounds are much moreflexible and stable to pressure changes.

Solutions of lipids or gaseous precursor-filled liposomes may bestabilized, for example, by the addition of a wide variety of viscositymodifiers, including, but not limited to carbohydrates and theirphosphorylated and sulfonated derivatives; polyethers, preferably withmolecular weight ranges between 400 and 8000; di- and trihydroxy alkanesand their polymers, preferably with molecular weight ranges between 800and 8000. Glycerol propylene glycol, polyethylene glycol, polyvinylpyrrolidone, and polyvinyl alcohol may also be useful as stabilizers inthe present invention. Particles which are porous or semi-solid such ashydroxyapatite, metal oxides and coprecipitates of gels, e.g. hyaluronicacid with calcium may be used to formulate a center or nidus tostabilize the gaseous precursors.

Emulsifying and/or solubilizing agents may also be used in conjunctionwith lipids or liposomes. Such agents include, but are not limited to,acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolinalcohols, lecithin, mono- and di-glycerides, mono-ethanolamine, oleicacid, oleyl alcohol, poloxamer, polyoxyethylene 50 stearate, polyoxyl 35castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether,polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60,polysorbate 80, propylene glycol diacetate, propylene glycolmonostearate, sodium lauryl sulfate, sodium stearate, sorbitanmono-laurate, sorbitan mono-oleate, sorbitan mono-palmitate, sorbitanmonostearate, stearic acid, trolamine, and emulsifying wax. All lipidswith perfluoro fatty acids as a component of the lipid in lieu of thesaturated or unsaturated hydrocarbon fatty acids found in lipids ofplant or animal origin may be used. Suspending and/orviscosity-increasing agents that may be used with lipid or liposomesolutions include but are not limited to, acacia, agar, alginic acid,aluminum mono-stearate, bentonite, magma, carbomer 934P,carboxymethylcellulose, calcium and sodium and sodium 12, glycerol,carrageenan, cellulose, dextrin, gelatin, guar gum, hydroxyethylcellulose, hydroxypropyl methylcellulose, magnesium aluminum silicate,methylcellulose, pectin, polyethylene oxide, polyvinyl alcohol,povidone, propylene glycol, alginate, silicon dioxide, sodium alginate,tragacanth, and xanthum gum. A preferred product of the presentinvention incorporates lipid as a mixed solvent system in a ratio of8:1:1 or 9:1:1 normal saline: glycerol:propylene glycol.

The gaseous precursor-filled liposomes of the present invention arepreferably comprised of an impermeable material. Impermeable material isdefined a material that does not permit the passage of a substantialamount of the contents of the liposome in typical storage conditions.Substantial is defined as greater than about 50% of the contents, thecontents being both the gas as well as any other component encapsulatedwithin the interior of the liposome, such as a therapeutic. Preferably,no more than about 25% of the gas is released, more preferably, no morethan about 10% of the gas is released, and most preferably, no more thanabout 1% of the gas is released during storage and prior toadministration to a patient.

At least in part, the gas impermeability of gaseous precursor-filledliposomes has been found to be related to the gel state to liquidcrystalline state phase transition temperature. It is believed that,generally, the higher gel state to liquid crystalline state phasetransition temperature, the more gas impermeable the liposomes are at agiven temperature. See Table I above and Derek Marsh, CRC Handbook ofLipid Bilayers (CRC Press, Boca Raton, Fla. 1990), at p. 139 for mainchain melting transitions of saturateddiacyl-sn-glycero-3-phosphocholines. However, it should be noted that alesser degree of energy can generally be used to release a therapeuticcompound from gaseous precursor-filled liposomes composed of lipids witha lower gel state to liquid crystalline state phase transitiontemperature.

In certain preferred embodiments, the phase transition temperature ofthe lipid is greater than the internal body temperature of the patientto which they are administered. For example, lipids having a phasetransition temperature greater than about 37° C. are preferred foradministration to humans. In general, microspheres having a gel toliquid phase transition temperature greater than about 20° C. areadequate and those with a phase transition temperature greater thanabout 37° C. are preferred.

In preferred embodiments, the liposomes made by the methods of thepresent invention are stable, stability being defined as resistance torupture from the time of formation until the application of ultrasound.The lipids used to construct the microspheres may be chosen forstability. For example, gaseous precursor-filled liposomes composed ofDSPC (distearoylphosphatidylcholine) are more stable than gaseousprecursor-filled liposomes composed of DPPC(dipalmitoylphosphatidylcholine) and that these in turn are more stablethan gaseous precursor-filled liposomes composed of eggphosphatidylcholine (EPC). Preferably, no more than about 50% of theliposomes rupture from the time of formation until the application ofultrasound, more preferably, no more than about 25% of the liposomesrupture, even more preferably, no more than about 10% of the liposomes,and most preferably, no more than about 1% of the liposomes.

The subject liposomes tend to have greater gas impermeability andstability during storage than other gas-filled liposomes produced viaknown procedures such as pressurization or other techniques. At 72 hoursafter formation, for example, conventionally prepared liposomes oftenare essentially devoid of gas, the gas having diffused out of theliposomes and/or the liposomes having ruptured and/or fused, resultingin a concomitant loss in reflectivity. In comparison, gaseousprecursor-filled liposomes of the present invention maintained inaqueous solution generally have a shelf life stability of greater thanabout three weeks, preferably a shelf life stability of greater thanabout four weeks, more preferably a shelf life stability of greater thanabout five weeks, even more preferably a shelf life stability of greaterthan about three months, and often a shelf life stability that is evenmuch longer, such as over six months, twelve months, or even two years.

In addition, it has been found that the gaseous precursor-filledliposomes of the present invention can be stabilized with lipidscovalently linked to polymers of polyethylene glycol, commonly referredto as PEGylated lipids. It has also been found that the incorporation ofat least a small amount of negatively charged lipid into any liposomemembrane, although not required, is beneficial to providing liposomesthat do not have a propensity to rupture by aggregation. By at least asmall amount, it is meant about 1 to about 10 mole percent of the totallipid. Suitable negatively charged lipids, or lipids bearing a netnegative charge, will be readily apparent to those skilled in the art,and include, for example, phosphatidylserine, phosphatidylglycerol,phosphatidic acid, and fatty acids. Liposomes prepared fromdipalmitoylphosphatidylcholine are most preferred as they are selectedfor their ability to rupture on application of resonant frequencyultrasound, radiofrequency energy, (e.g. microwave), and/or echogenicityin addition to their stability during delivery.

Further, the liposomes of the invention are preferably sufficientlystable in the vasculature such that they withstand recirculation. Thegaseous precursor-filled liposomes may be coated such that uptake by thereticuloendothelial system is minimized. Useful coatings include, forexample, gangliosides, glucuronide, galacturonate, guluronate,polyethyleneglycol, polypropylene glycol, polyvinylpyrrolidone,polyvinylalcohol, dextran, starch, phosphorylated and sulfonated mono,di, tri, oligo and polysaccharides and albumin. The liposomes may alsobe coated for purposes such as evading recognition by the immune system.

The lipid used is also preferably flexible. Flexibility, as defined inthe context of gaseous precursor-filled liposomes, is the ability of astructure to alter its shape, for example, in order to pass through anopening having a size smaller than the liposome.

Provided that the circulation half-life of the liposomes is sufficientlylong, the liposomes will generally pass through the target tissue whilepassing through the body. Thus, by focusing the sound waves on theselected tissue to be treated, the therapeutic will be released locallyin the target tissue. As a further aid to targeting, antibodies,carbohydrates, peptides, glycopeptides, glycolipids, lectins, andsynthetic and natural polymers may also be incorporated into the surfaceof the liposomes. Other aids for targeting include polymers such aspolyethyleneglycol, polyvinylpyrrolidone, and polvinylalcohol, which maybe incorporated onto the surface via alkylation, acylation, sterolgroups or derivatized head groups of phospholipids such asdioleoylphosphatidylethanolamine (DOPE),dipalmitoylphosphatidylethanolamine (DPPE), ordistearoylphosphatidylethanolamine (DSPE). Peptides, antibodies,lectins, glycopeptides, oligonucleotides, and glycoconjugates may alsobe incorporated onto the surfaces of the gaseous precursor-filled lipidspheres.

In certain preferred embodiments, as an aid to the gaseous precursorinstillation process as well as to maintain the stability of the gaseousprecursor-filled liposomes, for example, emulsifiers may be added to thelipid. Examples of emulsifiers include, but are not limited to,glycerol, cetyl alcohol, sorbitol, polyvinyl alcohol, polypropyleneglycol, propylene glycol, ethyl alcohol, sodium lauryl sulfate, Laureth23, polysorbates (all units), all saturated and unsaturated fatty acids,triethanolamine, Tween 20, tween 40, Tween 60, tween 80, Polysorbate 20,Polysorbate 40, Polysorbate 60, and Polysorbate 80.

For storage prior to use, the liposomes of the present invention may besuspended in an aqueous solution, such as a saline solution (forexample, a phosphate buffered saline solution), or simply water, andstored preferably at a temperature of between about 2° C. and about 10°C., preferably at about 4° C. Preferably, the water is sterile.

Typical storage conditions are, for example, a non-degassed aqueoussolution of 0.9% NaCl maintained at 4° C. for 48 hours. The temperatureof storage is preferably below the gel state to liquid crystalline statephase transition temperature of the material forming the liposomes.

Most preferably, the liposomes are stored in an isotonic salinesolution, although, if desired, the saline solution may be hypotonic(e.g., about 0.3 to about 0.5% NaCl). The solution also may be buffered,if desired, to provide a pH range of about pH 5 to about pH 7.4.Suitable buffers include, but are not limited to, acetate, citrate,phosphate, bicarbonate, and phosphate-buffered saline, 5% dextrose, andphysiological saline (normal saline).

Bacteriostatic agents may also be included with the liposomes to preventbacterial degradation on storage. Suitable bacteriostatic agents includebut are not limited to benzalkonium chloride, benzethonium chloride,benzoic acid, benzyl alcohol, butylparaben, cetylpyridinium chloride,chlorobutanol, chlorocresol, methylparaben, phenol, potassium benzoate,potassium sorbate, sodium benzoate and sorbic acid.

By "gas-filled" as used herein it is meant liposomes having an interiorvolume that is at least about 10% gas, preferably at least about 25%gas, more preferably at least about 50% gas, even more preferably atleast about 75% gas, and most preferably at least about 90% gas. It willbe understood by one skilled in the art, once armed with the presentdisclosure, that a gaseous precursor may also be used, followed byactivation to form a gas.

Various biocompatible gases may be employed in the gas-filled liposomesof the present invention. Such gases include air, nitrogen, carbondioxide, oxygen, argon, fluorine, xenon, neon, helium, or any and allcombinations thereof. Other suitable gases will be apparent to thoseskilled in the art once armed with the present disclosure. In additionto the gaseous precursors disclosed herein, the precursors may beco-entrapped with other gases. For example, during the transition fromthe gaseous precursor to a gas in an enclosed environment containingambient gas (as air), the two gases may mix and upon agitation andformation of microspheres, the gaseous content of the microspheresresults in a mixture of two or more gases, dependent upon the densitiesof the gases mixed.

The size of the liposomes of the present invention will depend upon theintended use. With the smaller liposomes, resonant frequency ultrasoundwill generally be higher than for the larger liposomes. Sizing alsoserves to modulate resultant liposomal biodistribution and clearance. Inaddition to filtration, the size of the liposomes can be adjusted, ifdesired, by procedures known to one skilled in the art, such asextrusion, sonication, homogenization, the use of a laminar stream of acore of liquid introduced into an immiscible sheath of liquid. See, forexample, U.S. Pat. No. 4,728,578; U.K. Patent Application GB 2193095 A;U.S. Pat. No. 4,728,575; U.S. Pat. No. 4,737,323; InternationalApplication PCT/US85/01161; Mayer et al., Biochimica et Biophysica Acta1986, 858, 161-168; Hope et al., Biochimica et Biophysica Acta 1985,812, 55-65; U.S. Pat. No. 4,533,254; Mayhew et al., Methods inEnzymology 1987, 149, 64-77; Mayhew et al., Biochimica et BiophysicaActa 1984, 755, 169-74; Cheng et al, Investigative Radiology 1987, 22,47-55; PCT/US89/05040; U.S. Pat. No. 4,162,282; U.S. Pat. No. 4,310,505;U.S. Pat. No. 4,921,706; and Liposomes Technology, Gregoriadis, G., ed.,Vol. I, pp. 29-37, 51-67 and 79-108 (CRC Press Inc, Boca Raton, Fla.,1984). The disclosures of each of the foregoing patents, publicationsand patent applications are incorporated by reference herein, in theirentirety. Extrusion under pressure through pores of defined size is apreferred method of adjusting the size of the liposomes.

Since liposome size influences biodistribution, different size liposomesmay be selected for various purposes. For example, for intravascularapplication, the preferred size range is a mean outside diameter betweenabout 30 nanometers and about 10 microns, with the preferable meanoutside diameter being about 5 microns.

More specifically, for intravascular application, the size of theliposomes is preferably about 10 μm or less in mean outside diameter,and preferably less than about 7 μm, and more preferably no smaller thanabout 5 nanometers in mean outside diameter. Preferably, the liposomesare no smaller than about 30 nanometers in mean outside diameter.

To provide therapeutic delivery to organs such as the liver and to allowdifferentiation of tumor from normal tissue, smaller liposomes, betweenabout 30 nanometers and about 100 nanometers in mean outside diameter,are preferred.

For embolization of a tissue such as the kidney or the lung, theliposomes are preferably less than about 200 microns in mean outsidediameter.

For intranasal, intrarectal or topical administration, the microspheresare preferably less than about 100 microns in mean outside diameter.

Large liposomes, e.g., between 1 and 10 microns in size, will generallybe confined to the intravascular space until they are cleared byphagocytic elements lining the vessels, such as the macrophages andKuppfer cells lining capillary sinusoids. For passage to the cellsbeyond the sinusoids, smaller liposomes, for example, less than about amicron in mean outside diameter, e.g., less than about 300 nanometers insize, may be utilized.

The route of administration of the liposomes will vary depending on theintended use. As one skilled in the art would recognize, administrationof therapeutic delivery systems of the present invention may be carriedout in various fashions, such as intravascularly, intralymphatically,parenterally, subcutaneously, intramuscularly, intranasally,intrarectally, intraperitoneally, interstitially, into the airways vianebulizer, hyperbarically, orally, topically, or intratumorly, using avariety of dosage forms. One preferred route of administration isintravascularly. For intravascular use, the therapeutic delivery systemis generally injected intravenously, but may be injected intraarteriallyas well. The liposomes of the invention may also be injectedinterstitially or into any body cavity.

The delivery of therapeutics from the liposomes of the present inventionusing ultrasound is best accomplished for tissues which have a goodacoustic window for the transmission of ultrasonic energy. This is thecase for most tissues in the body such as muscle, the heart, the liverand most other vital structures. In the brain, in order to direct theultrasonic energy past the skull a surgical window may be necessary. Forbody parts without an acoustic window, e.g. through bone, radiofrequencyor microwave energy is preferred.

Additionally, the invention is especially useful in deliveringtherapeutics to a patient's lungs. Gaseous precursor-filled liposomes ofthe present invention are lighter than, for example, conventionalliquid-filled liposomes which generally deposit in the central proximalairway rather than reaching the periphery of the lungs. It is thereforebelieved that the gaseous precursor-filled liposomes of the presentinvention may improve delivery of a therapeutic compound to theperiphery of the lungs, including the terminal airways and the alveoli.For application to the lungs, the gaseous precursor-filled liposomes maybe applied through nebulization, for example.

2 cc of liposomes (lipid=83% DPPC/8% DPPE-PEG 5,000/5% DPPA) entrappingair was placed in a nebulizer and nebulized. The resulting liposomespost nebulization, were around 1 to 2 microns in size and were shown tofloat in the air. These size particles appear ideal for deliveringdrugs, peptides, genetic materials and other therapeutic compounds intothe far reaches of the lung (i.e. terminal airways and alveoli). Becausethe gas filled liposomes are almost as light as air, much lighter thanconventional water filled liposomes, they float longer in the air, andas such are better for delivering compounds into the distal lung. WhenDNA is added to these liposomes, it is readily adsorbed or bound to theliposomes. Thus, liposomes and microspheres filled by gas and gaseousprecursors hold vast potential for pulmonary drug delivery.

In applications such as the targeting of the lungs, which are lined withlipids, the therapeutic may be released upon aggregation of the gaseousprecursor-filled liposome with the lipids lining the targeted tissue.Additionally, the gaseous precursor-filled liposomes may burst afteradministration without the use of ultrasound. Thus, ultrasound need notbe applied to release the drug in the above type of administration.

Further, the gaseous precursor-filled liposomes of the invention areespecially useful for therapeutics that may be degraded in aqueous mediaor upon exposure to oxygen and/or atmospheric air. For example, theliposomes may be filled with an inert gas such as nitrogen or argon, foruse with labile therapeutic compounds. Additionally, the gaseousprecursor-filled liposomes may be filled with an inert gas and used toencapsulate a labile therapeutic for use in a region of a patient thatwould normally cause the therapeutic to be exposed to atmospheric air,such as cutaneous and ophthalmic applications.

The gaseous precursor-filled liposomes are also especially useful fortranscutaneous delivery, such as a patch delivery system. The use ofrupturing ultrasound may increase transdermal delivery of therapeuticcompounds. Further, a mechanism may be used to monitor and modulate drugdelivery. For example, diagnostic ultrasound may be used to visuallymonitor the bursting of the gaseous precursor-filled liposomes andmodulate drug delivery and/or a hydrophone may be used to detect thesound of the bursting of the gaseous precursor-filled liposomes andmodulate drug delivery.

In preferred embodiments, the gas-filled liposomes are administered in avehicle as individual particles, as opposed to being embedded in apolymeric matrix for the purposes of controlled release.

For in vitro use, such as cell culture applications, the gaseousprecursor-filled liposomes may be added to the cells in cultures andthen incubated. Subsequently sonic energy, microwave, or thermal energy(e.g. simple heating) can be applied to the culture media containing thecells and liposomes.

Generally, the therapeutic delivery systems of the invention areadministered in the form of an aqueous suspension such as in water or asaline solution (e.g., phosphate buffered saline). Preferably, the wateris sterile. Also, preferably the saline solution is an isotonic salinesolution, although, if desired, the saline solution may be hypotonic(e.g., about 0.3 to about 0.5% NaCl). The solution may also be buffered,if desired, to provide a pH range of about pH 5 to about pH 7.4. Inaddition, dextrose may be preferably included in the media. Furthersolutions that may be used for administration of gaseousprecursor-filled liposomes include, but are not limited to almond oil,corn oil, cottonseed oil, ethyl oleate, isopropyl myristate, isopropylpalmirate, mineral oil, myristyl alcohol, octyldodecanol, olive oil,peanut oil, persic oil, sesame oil, soybean oil, squalene, myristyloleate, cetyl oleate, myristyl palmitate, as well as other saturated andunsaturated alkyl chain alcohols (C=2-22) esterified to alkyl chainfatty acids (C=2-22).

The useful dosage of gaseous precursor-filled microspheres to beadministered and the mode of administration will vary depending upon theage, weight, and mammal to be treated, and the particular application(therapeutic/diagnostic) intended. Typically, dosage is initiated atlower levels and increased until the desired therapeutic effect isachieved.

For use in ultrasonic imaging, preferably, the liposomes of theinvention possess a reflectivity of greater than 2 dB, more preferablybetween about 4 dB and about 20 dB. Within these ranges, the highestreflectivity for the liposomes of the invention is exhibited by thelarger liposomes, by higher concentrations of liposomes, and/or whenhigher ultrasound frequencies are employed.

For therapeutic drug delivery, the rupturing of the therapeuticcontaining liposomes of the invention is surprisingly easily carried outby applying ultrasound of a certain frequency to the region of thepatient where therapy is desired, after the liposomes have beenadministered to or have otherwise reached that region. Specifically, ithas been unexpectedly found that when ultrasound is applied at afrequency corresponding to the peak resonant frequency of thetherapeutic containing gaseous precursor-filled liposomes, the liposomeswill rupture and release their contents.

The peak resonant frequency can be determined either in vivo or invitro, but preferably in vivo, by exposing the liposomes to ultrasound,receiving the reflected resonant frequency signals and analyzing thespectrum of signals received to determine the peak, using conventionalmeans. The peak, as so determined, corresponds to the peak resonantfrequency (or second harmonic, as it is sometimes termed).

Preferably, the liposomes of the invention have a peak resonantfrequency of between about 0.5 mHz and about 10 mHz. Of course, the peakresonant frequency of the gaseous precursor-filled liposomes of theinvention will vary depending on the outside diameter and, to someextent, the elasticity or flexibility of the liposomes, with the largerand more elastic or flexible liposomes having a lower resonant frequencythan the smaller and less elastic or flexible liposomes.

The therapeutic-containing gaseous precursor-filled liposomes will alsorupture when exposed to non-peak resonant frequency ultrasound incombination with a higher intensity (wattage) and duration (time). Thishigher energy, however, results in greatly increased heating, which maynot be desirable. By adjusting the frequency of the energy to match thepeak resonant frequency, the efficiency of rupture and therapeuticrelease is improved, appreciable tissue heating does not generally occur(frequently no increase in temperature above about 2° C.), and lessoverall energy is required. Thus, application of ultrasound at the peakresonant frequency, while not required, is most preferred.

For diagnostic or therapeutic ultrasound, any of the various types ofdiagnostic ultrasound imaging devices may be employed in the practice ofthe invention, the particular type or model of the device not beingcritical to the method of the invention. Also suitable are devicesdesigned for administering ultrasonic hyperthermia, such devices beingdescribed in U.S. Pat. Nos. 4,620,546, 4,658,828, and 4,586,512, thedisclosures of each of which are hereby incorporated herein by referencein their entirety. Preferably, the device employs a resonant frequency(RF) spectral analyzer. The transducer probes may be applied externallyor may be implanted. Ultrasound is generally initiated at lowerintensity and duration, and then intensity, time, and/or resonantfrequency increased until the liposome is visualized on ultrasound (fordiagnostic ultrasound applications) or ruptures (for therapeuticultrasound applications).

Although application of the various principles will be readily apparentto one skilled in the art, once armed with the present disclosure, byway of general guidance, for gaseous precursor-filled liposomes of about1.5 to about 10 microns in mean outside diameter, the resonant frequencywill generally be in the range of about 1 to about 10 megahertz. Byadjusting the focal zone to the center of the target tissue (e.g., thetumor) the gaseous precursor-filled liposomes can be visualized underreal time ultrasound as they accumulate within the target tissue. Usingthe 7.5 megahertz curved array transducer as an example, adjusting thepower delivered to the transducer to maximum and adjusting the focalzone within the target tissue, the spatial peak temporal average (SPTA)power will then be a maximum of approximately 5.31 mW/cm² in water. Thispower will cause some release of therapeutic from the gaseousprecursor-filled liposomes, but much greater release can be accomplishedby using higher power.

By switching the transducer to the doppler mode, higher power outputsare available, up to 2.5 watts per cm² from the same transducer. Withthe machine operating in doppler mode, the power can be delivered to aselected focal zone within the target tissue and the gaseousprecursor-filled liposomes can be made to release their therapeutics.Selecting the transducer to match the resonant frequency of the gaseousprecursor-filled liposomes will make this process of therapeutic releaseeven more efficient.

For larger diameter gaseous precursor-filled liposomes, e.g., greaterthan 3 microns in mean outside diameter, a lower frequency transducermay be more effective in accomplishing therapeutic release. For example,a lower frequency transducer of 3.5 megahertz (20 mm curved array model)may be selected to correspond to the resonant frequency of the gaseousprecursor-filled liposomes. Using this transducer, 101.6 milliwatts percm² may be delivered to the focal spot, and switching to doppler modewill increase the power output (SPTA) to 1.02 watts per cm².

To use the phenomenon of cavitation to release and/or activate thedrugs/prodrugs within the gaseous precursor-filled liposomes, lowerfrequency energies may be used, as cavitation occurs more effectively atlower frequencies. Using a 0.757 megahertz transducer driven with highervoltages (as high as 300 volts) cavitation of solutions of gaseousprecursor-filled liposomes will occur at thresholds of about 5.2atmospheres.

Table III shows the ranges of energies transmitted to tissues fromdiagnostic ultrasound on commonly used instruments such as the PiconicsInc. (Tyngsboro, Mass.) Portascan general purpose scanner with receiverpulser 1966 Model 661; the Picker (Cleveland, Ohio) Echoview 8L Scannerincluding 80C System or the Medisonics (Mountain View, Calif.) Model D-9Versatone Bidirectional Doppler. In general, these ranges of energiesemployed in pulse repetition are useful for diagnosis and monitoring thegas-filled liposomes but are insufficient to rupture the gas-filledliposomes of the present invention.

                  TABLE III                                                       ______________________________________                                        Power and Intensities Produced by Diagnostic Equipment*                                                  Average Intensity                                  Pulse repetition                                                                         Total ultrasonic                                                                              at transducer face                                 rate (Hz)  power output P (mW)                                                                           I.sub.TD (W/m.sup.2)                               ______________________________________                                        520        4.2             32                                                 676        9.4             71                                                 806        6.8             24                                                 1000       14.4            51                                                 1538       2.4             8.5                                                ______________________________________                                         *Values obtained from Carson et al., Ultrasound in Med. & Biol. 1978, 3,      341-350, the disclosures of which are hereby incorporated herein by           reference in their entirety.                                             

Higher energy ultrasound such as commonly employed in therapeuticultrasound equipment is preferred for activation of the therapeuticcontaining gaseous precursor-filled liposomes. In general, therapeuticultrasound machines employ as much as 50% to 100% duty cycles dependentupon the area of tissue to be heated by ultrasound. Areas with largeramounts of muscle mass (i.e., backs, thighs) and highly vascularizedtissues such as heart may require the larger duty cycle, e.g., 100%.

In diagnostic ultrasound, one or several pulses of sound are used andthe machine pauses between pulses to receive the reflected sonicsignals. The limited number of pulses used in diagnostic ultrasoundlimits the effective energy which is delivered to the tissue which isbeing imaged.

In therapeutic ultrasound, continuous wave ultrasound is used to deliverhigher energy levels. In using the liposomes of the present invention,the sound energy may be pulsed, but continuous wave ultrasound ispreferred. If pulsing is employed, the sound will preferably be pulsedin echo train lengths of at least about 8 and preferably at least about20 pulses at a time.

Either fixed frequency or modulated frequency ultrasound may be used.Fixed frequency is defined wherein the frequency of the sound wave isconstant over time. A modulated frequency is one in which the wavefrequency changes over time, for example, from high to low (PRICH) orfrom low to high (CHIRP). For example, a PRICH pulse with an initialfrequency of 10 MHz of sonic energy is swept to 1 MHz with increasingpower from 1 to 5 watts. Focused, frequency modulated, high energyultrasound may increase the rate of local gaseous expansion within theliposomes and rupturing to provide local delivery of therapeutics.

The frequency of the sound used may vary from about 0.025 to about 100megahertz. Frequency ranges between about 0.75 and about 3 megahertz arepreferred and frequencies between about 1 and about 2 megahertz are mostpreferred. Commonly used therapeutic frequencies of about 0.75 to about1.5 megahertz may be used. Commonly used diagnostic frequencies of about3 to about 7.5 megahertz may also be used. For very small liposomes,e.g., below 0.5 micron in mean outside diameter, higher frequencies ofsound may be preferred as these smaller liposomes will absorb sonicenergy more effectively at higher frequencies of sound. When very highfrequencies are used, e.g., over 10 megahertz, the sonic energy willgenerally have limited depth penetration into fluids and tissues.External application may be preferred for the skin and other superficialtissues, but for deep structures, the application of sonic energy viainterstitial probes or intravascular ultrasound catheters may bepreferred.

Where the gaseous precursor-filled liposomes are used for therapeuticdelivery, the therapeutic compound to be delivered may be embeddedwithin the wall of the liposome, encapsulated in the liposome and/orattached to the liposome, as desired. The phrase "attached to" orvariations thereof, as used herein in connection with the location ofthe therapeutic compound, means that the therapeutic compound is linkedin some manner to the inside and/or the outside wall of the microsphere,such as through a covalent or ionic bond or other means of chemical orelectrochemical linkage or interaction. The phrase "encapsulated invariations thereof" as used in connection with the location of thetherapeutic compound denotes that the therapeutic compound is located inthe internal microsphere void. The phrase "embedded within" orvariations thereof as used in connection with the location of thetherapeutic compound, signifies the positioning of the therapeuticcompound within the microsphere wall. The phrase "comprising atherapeutic" denotes all of the varying types of therapeutic positioningin connection with the microsphere. Thus, the therapeutic can bepositioned variably, such as, for example, entrapped within the internalvoid of the gaseous precursor-filled microsphere, situated between thegaseous precursor and the internal wall of the gaseous precursor-filledmicrosphere, incorporated onto the external surface of the gaseousprecursor-filled microsphere and/or enmeshed within the microspherestructure itself.

Any of a variety of therapeutics may be encapsulated in the liposomes.By therapeutic, as used herein, it is meant an agent having beneficialeffect on the patient. As used herein, the term therapeutic issynonymous with the terms contrast agent and/or drug.

Examples of drugs that may be delivered with gaseous precursor-filledliposomes may contain for drug delivery purposes, but by no means islimited to; hormone products such as, vasopressin and oxytocin and theirderivatives, glucagon, and thyroid agents as iodine products andanti-thyroid agents; cardiovascular products as chelating agents andmercurial diuretics and cardiac glycosides; respiratory products asxanthine derivatives (theophylline & aminophylline); anti-infectives asaminoglycosides, antifungals (amphotericin), penicillin andcephalosporin antibiotics, antiviral agents as Zidovudine, Ribavirin,Amantadine, Vidarabine, and Acyclovir, anti-helmintics, antimalarials,and antituberculous drugs; biologicals as immune serums, antitoxins andantivenins, rabies prophylaxis products, bacterial vaccines, viralvaccines, toxoids; antineoplastics as nitrosureas, nitrogen mustards,antimetabolites (fluorouracil, hormones as progestins and estrogens andantiestrogens; antibiotics as Dactinomycin; mitotic inhibitors asEtoposide and the Vinca alkaloids, Radiopharmaceuticals as radioactiveiodine and phosphorus products; as well as Interferon, hydroxyurea,procarbazine, Dacarbazine, Mitotane, Asparaginase and cyclosporins.

Genetic and bioactive materials may be incorporated into the internalgaseous precursor-filled space of these liposomes during the gaseousprecursor installation process or into or onto the lipid membranes ofthese particles. Incorporation onto the surface of these particles ispreferred. Genetic materials and bioactive products with a highoctanol/water partition coefficient may be incorporated directly intothe lipid layer surrounding the gaseous precursor but incorporation ontothe surface of the gaseous precursor-filled lipid spheres is morepreferred. To accomplish this, groups capable of binding geneticmaterials or bioactive materials are generally incorporated into thelipid layers which will then bind these materials. In the case ofgenetic materials (DNA, RNA, both single stranded and double strandedand antisense and sense oligonucleotides) this is readily accomplishedthrough the use of cationic lipids or cationic polymers which may beincorporated into the dried lipid starting materials.

It is the surprising discovery of the invention that liposomes,gas-filled and gas precursor-filled, when produced with phosphatidicacid, e.g. dipalmitoylphosphatidic acid in molar amounts in excess of 5mole % and preferably about 10 mole %, function as highly effectivebinders of genetic material. Such liposomes bind DNA avidly. This issurprising since positively charged liposomes were heretofore recognizedas most useful for binding DNA. Liposomes with 5 mole % to 10 mole %DPPA function as highly effective gas and gaseous precursor retainingstructures. Compositions incorporating phosphatidic acid are more robustfor diagnostic ultrasound and useful for carrying DNA as well as otherpharmaceuticals.

It is believed that nanoparticles, microparticles, and emulsions ofcertain precursors are particularly effective at accumulating inischemic and diseased tissue. Such precursors can be used for detectingischemic and diseased tissue via ultrasound and also for deliveringdrugs to these tissues. By co-entrapping drugs with the emulsions ornanoparticles comprising the gaseous precursors said drugs can then bedelivered to the diseased tissues. For example, emulsions of, sulfurhexafluoride, hesafluoropropylene, bromochlorofluoromethane,octafluoropropane, 1,1dichloro,fluoro ethane, hexafluoroethane,hesafluoro-2-butyne, perfluoropentane, perfluorobutane,octafluoro-2-butene or hexafluorobuta-1,3-diene oroctafluorocyclopentene (27° C.) can be used to deliver drugs such ascardiac glycosides, angiogenic factors and vasoactive compounds toischemic regions of the myocardium. Similarly, emulsions of the aboveprecursors may also be used to deliver antisense DNA orchemotherapeutics to tumors. It is postulated that subtle changes intemperature, pH and oxygen tension are responsible for the accumulationof certain precursors preferentially by diseased and ischemic tissues.These precursors can be used as a delivery vehicle or in ultrasound fordrug delivery.

Suitable therapeutics include, but are not limited to paramagneticgases, such as atmospheric air, which contains traces of oxygen 17;paramagnetic ions such as Mn⁺², Gd⁺², Fe⁺³ ; iron oxides or magnetite(Fe₃ O₄) and may thus be used as susceptibility contrast agents formagnetic resonance imaging (MRI), radioopaque metal ions, such asiodine, barium, bromine, or tungsten, for use as x-ray contrast agents,gases from quadrupolar nuclei, may have potential for use as MagneticResonance contrast agents, antineoplastic agents, such as platinumcompounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate,fluorouracil, adriamycin, taxol, mitomycin, ansamitocin, bleomycin,cytosine arabinoside, arabinosyl adenine, mercaptopolylysine,vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L-PAM orphenylalanine mustard), mercaptopurine, mitotane, procarbazinehydrochloride dactinomycin (actinomycin D), daunorubicin hydrochloride,doxorubicin hydrochloride, mitomycin, plicamycin (mithramycin),aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolideacetate, megestrol acetate, tamoxifen citrate, testolactone, trilostane,amsacrine (m-AMSA), asparaginase (L-asparaginase) Erwina asparaginase,etoposide (VP-16), interferon α-2a, interferon α-2b, teniposide (VM-26),vinblastine sulfate (VLB), vincristine sulfate, bleomycin, bleomycinsulfate, methotrexate, adriamycin, and arabinosyl, hydroxyurea,procarbazine, and dacarbazine; mitotic inhibitors such as etoposide andthe vinca alkaloids, radiopharmaceuticals such as radioactive iodine andphosphorus products; hormones such as progestins, estrogens andantiestrogens; anti-helmintics, antimalarials, and antituberculosisdrugs; biologicals such as immune serums, antitoxins and antivenins;rabies prophylaxis products; bacterial vaccines; viral vaccines;aminoglycosides; respiratory products such as xanthine derivativestheophylline and aminophylline; thyroid agents such as iodine productsand anti-thyroid agents; cardiovascular products including chelatingagents and mercurial diuretics and cardiac glycosides; glucagon; bloodproducts such as parenteral iron, hemin, hematoporphyrins and theirderivatives; biological response modifiers such as muramyldipeptide,muramyltripeptide, microbial cell wall components, lymphokines (e.g.,bacterial endotoxin such as lipopolysaccharide, macrophage activationfactor), sub-units of bacteria (such as Mycobacteria, Corynebacteria),the synthetic dipeptide N-acetyl-muramyl-L-alanyl-D-isoglutamine;anti-fungal agents such as ketoconazole, nystatin, griseofulvin,flucytosine (5-FC), miconazole, amphotericin B, ricin, cyclosporins, andβ-lactam antibiotics (e.g., sulfazecin); hormones such as growthhormone, melanocyte stimulating hormone, estradiol, beclomethasonedipropionate, betamethasone, betamethasone acetate and betamethasonesodium phosphate, vetamethasone disodium phosphate, vetamethasone sodiumphosphate, cortisone acetate, dexamethasone, dexamethasone acetate,dexamethasone sodium phosphate, flunsolide, hydrocortisone,hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodiumphosphate, hydrocortisone sodium succinate, methylprednisolone,methylprednisolone acetate, methylprednisolone sodium succinate,paramethasone acetate, prednisolone, prednisolone acetate, prednisolonesodium phosphate, prednisolone terbutate, prednisone, triamcinolone,triamcinolone acetonide, triamcinolone diacetate, triamcinolonehexacetonide and fludrocortisone acetate, oxytocin, vassopressin, andtheir derivatives; vitamins such as cyanocobalamin neinoic acid,retinoids and derivatives such as retinol palmitate, and α-tocopherol;peptides, such as manganese super oxide dimutase; enzymes such asalkaline phosphatase; anti-allergic agents such as amelexanox;anti-coagulation agents such as phenprocoumon and heparin; circulatorydrugs such as propranolol; metabolic potentiators such as glutathione;antituberculars such as paraaminosalicylic acid, isoniazid, capreomycinsulfate cycloserine, ethambutol hydrochloride ethionamide, pyrazinamide,rifampin, and streptomycin sulfate; antivirals such as acyclovir,amantadine azidothymidine (AZT or Zidovudine), ribavirin and vidarabinemonohydrate (adenine arabinoside, ara-A); antianginals such asdiltiazem, nifedipine, verapamil, erythrityl tetranitrate, isosorbidedinitrate, nitroglycerin (glyceryl trinitrate) and pentaerythritoltetranitrate; anticoagulants such as phenprocoumon, heparin; antibioticssuch as dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil,cephalexin, cephradine erythromycin, clindamycin, lincomycin,amoxicillin, ampicillin, bacampicillin, carbenicillin, dicloxacillin,cyclacillin, picloxacillin, hetacillin, methicillin, nafcillin,oxacillin, penicillin, including penicillin G, penicillin V, ticarcillinrifampin and tetracycline; antiinflammatories such as difunisal,ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen,oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirinand salicylates; antiprotozoans such as chloroquine, hydroxychloroquine,metronidazole, quinine and meglumine antimonate; antirheumatics such aspenicillamine; narcotics such as paregoric; opiates such as codeine,heroin, methadone, morphine and opium; cardiac glycosides such asdeslanoside, digitoxin, digoxin, digitalin and digitalis; neuromuscularblockers such as atracurium besylate, gallamine triethiodide,hexafluorenium bromide, metocurine iodide, pancuronium bromide,succinylcholine chloride (suxamethonium chloride), tubocurarine chlorideand vecuronium bromide; sedatives (hypnotics) such as amobarbital,amobarbital sodium, aprobarbital, butabarbital sodium, chloral hydrate,ethchlorvynol, ethinamate, flurazepam hydrochloride, glutethimide,methotrimeprazine hydrochloride, methyprylon, midazolam hydrochloride,paraldehyde, pentobarbital, pentobarbital sodium, phenobarbital sodium,secobarbital sodium, talbutal, temazepam and triazolam; localanesthetics such as bupivacaine hydrochloride, chloroprocainehydrochloride, etidocaine hydrochloride, lidocaine hydrochloride,mepivacaine hydrochloride, procaine hydrochloride and tetracainehydrochloride; general anesthetics such as droperidol, etomidate,fentanyl citrate with droperidol, ketamine hydrochloride, methohexitalsodium and thiopental sodium; and radioactive particles or ions such asstrontium, iodide rhenium and yttrium.

In certain preferred embodiments, the therapeutic is a monoclonalantibody, such as a monoclonal antibody capable of binding to melanomaantigen.

Other preferred therapeutics include genetic material such as nucleicacids, RNA, and DNA, of either natural or synthetic origin, includingrecombinant RNA and DNA and antisense RNA and DNA. Types of geneticmaterial that may be used include, for example, genes carried onexpression vectors such as plasmids, phagemids, cosmids, yeastartificial chromosomes (YACs), and defective or "helper" viruses,antigene nucleic acids, both single and double stranded RNA and DNA andanalogs thereof, such as phosphorothioate, phosphoroamidate, andphosphorodithioate oligodeoxynucleotides. Additionally, the geneticmaterial may be combined, for example, with proteins or other polymers.

Examples of genetic therapeutics that may be applied using the liposomesof the present invention include DNA encoding at least a portion of anHLA gene, DNA encoding at least a portion of dystrophin, DNA encoding atleast a portion of CFTR, DNA encoding at least a portion of IL-2, DNAencoding at least a portion of TNF, an antisense oligonucleotide capableof binding the DNA encoding at least a portion of Ras.

DNA encoding certain proteins may be used in the treatment of manydifferent types of diseases. For example, adenosine deaminase may beprovided to treat ADA deficiency; tumor necrosis factor and/orinterleukin-2may be provided to treat advanced cancers; HDL receptor maybe provided to treat liver disease; thymidine kinase may be provided totreat ovarian cancer, brain tumors, or HIV infection; HLA-B7 may beprovided to treat malignant melanoma; interleukin-2 may be provided totreat neuroblastoma, malignant melanoma, or kidney cancer; interleukin-4may be provided to treat cancer; HIV env may be provided to treat HIVinfection; antisense ras/p53 may be provided to treat lung cancer; andFactor VIII may be provided to treat Hemophilia B. See, for example,Thompson, L., Science, 1992, 258, 744-746.

If desired, more than one therapeutic may be applied using theliposomes. For example, a single liposome may contain more than onetherapeutic or liposomes containing different therapeutics may beco-administered. By way of example, a monoclonal antibody capable ofbinding to melanoma antigen and an oligonucleotide encoding at least aportion of IL-2 may be administered at the same time. The phrase "atleast a portion of," as used herein, means that the entire gene need notbe represented by the oligonucleotide, so long as the portion of thegene represented provides an effective block to gene expression.

Similarly, prodrugs may be encapsulated in the liposomes, and areincluded within the ambit of the term therapeutic, as used herein.Prodrugs are well known in the art and include inactive drug precursorswhich, when exposed to high temperature, metabolizing enzymes,cavitation and/or pressure, in the presence of oxygen or otherwise, orwhen released from the liposomes, will form active drugs. Such prodrugscan be activated from, or released from, gas-filled lipid spheres in themethod of the invention, upon the application of ultrasound orradiofrequency microwave energy to the prodrug-containing liposomes withthe resultant cavitation, heating, pressure, and/or release from theliposomes. Suitable prodrugs will be apparent to those skilled in theart, and are described, for example, in Sinkula et al., J. Pharm. Sci.1975, 64, 181-210, the disclosure of which are hereby incorporatedherein by reference in its entirety.

Prodrugs, for example, may comprise inactive forms of the active drugswherein a chemical group is present on the prodrug which renders itinactive and/or confers solubility or some other property to the drug.In this form, the prodrugs are generally inactive, but once the chemicalgroup has been cleaved from the prodrug, by heat, cavitation, pressure,and/or by enzymes in the surrounding environment or otherwise, theactive drug is generated. Such prodrugs are well described in the art,and comprise a wide variety of drugs bound to chemical groups throughbonds such as esters to short, medium or long chain aliphaticcarbonates, hemiesters of organic phosphate, pyrophosphate, sulfate,amides, amino acids, azo bonds, carbamate, phosphamide, glucosiduronate,N-acetylglucosamine and β-glucoside.

Examples of drugs with the parent molecule and the reversiblemodification or linkage are as follows: convallatoxin with ketals,hydantoin with alkyl esters, chlorphenesin with glycine or alanineesters, acetaminophen with caffeine complex, acetylsalicylic acid withTHAM salt, acetylsalicylic acid with acetamidophenyl ester, naloxonewith sulfate ester, 15-methylprostaglandin F₂α with methyl ester,procaine with polyethylene glycol, erythromycin with alkyl esters,clindamycin with alkyl esters or phosphate esters, tetracycline withbetaine salts, 7-acylaminocephalosporins with ring-substitutedacyloxybenzyl esters, nandrolone with phenylproprionate decanoateesters, estradiol with enol ether acetal, methylprednisolone withacetate esters, testosterone with n-acetylglucosaminide glucosiduronate(trimethylsilyl) ether, cortisol or prednisolone or dexamethasone with21-phosphate esters.

Prodrugs may also be designed as reversible drug derivatives andutilized as modifiers to enhance drug transport to site-specifictissues. Examples of parent molecules with reversible modifications orlinkages to influence transport to a site specific tissue and forenhanced therapeutic effect include isocyanate with haloalkylnitrosurea, testosterone with propionate ester, methotrexate(3-5'-dichloromethotrexate) with dialkyl esters, cytosine arabinosidewith 5'-acylate, nitrogen mustard (2,2'-dichloro-N-methyldiethylamine),nitrogen mustard with aminomethyl tetracycline, nitrogen mustard withcholesterol or estradiol or dehydroepiandrosterone esters and nitrogenmustard with azobenzene.

As one skilled in the art would recognize, a particular chemical groupto modify a given drug may be selected to influence the partitioning ofthe drug into either the membrane or the internal space of theliposomes. The bond selected to link the chemical group to the drug maybe selected to have the desired rate of metabolism, e.g., hydrolysis inthe case of ester bonds in the presence of serum esterases after releasefrom the gaseous precursor-filled liposomes. Additionally, theparticular chemical group may be selected to influence thebiodistribution of the drug employed in the gaseous precursor-filleddrug carrying liposome invention, e.g.,N,N-bis(2-chloroethyl)phosphorodiamidic acid with cyclic phosphoramidefor ovarian adenocarcinoma.

Additionally, the prodrugs employed within the gaseous precursor-filledliposomes may be designed to contain reversible derivatives which areutilized as modifiers of duration of activity to provide, prolong ordepot action effects. For example, nicotinic acid may be modified withdextran and carboxymethlydextran esters, streptomycin with alginic acidsalt, dihydrostreptomycin with pamoate salt, cytarabine (ara-C) with5'-adamantoate ester, ara-adenosine (ara-A) with 5-palmitate and5'-benzoate esters, amphotericin B with methyl esters, testosterone with17-β-alkyl esters, estradiol with formate ester, prostaglandin with2-(4-imidazolyl)ethylamine salt, dopamine with amino acid amides,chloramphenicol with mono- and bis(trimethylsilyl) ethers, andcycloguanil with pamoate salt. In this form, a depot or reservoir oflong-acting drug may be released in vivo from the gaseousprecursor-filled prodrug bearing liposomes.

In addition, compounds which are generally thermally labile may beutilized to create toxic free radical compounds. Compounds withazolinkages, peroxides and disulfide linkages which decompose with hightemperature are preferred. With this form of prodrug, azo, peroxide ordisulfide bond containing compounds are activated by cavitation and/orincreased heating caused by the interaction of high energy sound withthe gaseous precursor-filled liposomes to create cascades of freeradicals from these prodrugs entrapped therein. A wide variety of drugsor chemicals may constitute these prodrugs, such as azo compounds, thegeneral structure of such compounds being R--N═N--R, wherein R is ahydrocarbon chain, where the double bond between the two nitrogen atomsmay react to create free radical products in vivo.

Exemplary drugs or compounds which may be used to create free radicalproducts include azo containing compounds such as azobenzene,2,2'-azobisisobutyronitrile, azodicarbonamide, azolitmin, azomycin,azosemide, azosulfamide, azoxybenzene, aztreonam, sudan III,sulfachrysoidine, sulfamidochrysoidine and sulfasalazine, compoundscontaining disulfide bonds such as sulbentine, thiamine disulfide,thiolutin, thiram, compounds containing peroxides such as hydrogenperoxide and benzoylperoxide, 2,2'-azobisisobutyronitrile,2,2'-azobis(2-amidopropane) dihydrochloride, and2,2'-azobis(2,4-dimethylvaleronitrile).

A gaseous precursor-filled liposome filled with oxygen gas should createextensive free radicals with cavitation. Also, metal ions from thetransition series, especially manganese, iron and copper can increasethe rate of formation of reactive oxygen intermediates from oxygen. Byencapsulating metal ions within the liposomes, the formation of freeradicals in vivo can be increased. These metal ions may be incorporatedinto the liposomes as free salts, as complexes, e.g., with EDTA, DTPA,DOTA or desferrioxamine, or as oxides of the metal ions. Additionally,derivatized complexes of the metal ions may be bound to lipid headgroups, or lipophilic complexes of the ions may be incorporated into alipid bilayer, for example. When exposed to thermal stimulation, e.g.,cavitation, these metal ions then will increase the rate of formation ofreactive oxygen intermediates. Further, radiosensitizers such asmetronidazole and misonidazole may be incorporated into the gaseousprecursor-filled liposomes to create free radicals on thermalstimulation.

By way of an example of the use of prodrugs, an acylated chemical groupmay be bound to a drug via an ester linkage which would readily cleavein vivo by enzymatic action in serum. The acylated prodrug isincorporated into the gaseous precursor-filled liposome of theinvention. The derivatives, in addition to hydrocarbon and substitutedhydrocarbon alkyl groups, may also be composed of halo substituted andperhalo substituted groups as perfluoroalkyl groups. Perfluoroalkylgroups should possess the ability to stabilize the emulsion. When thegaseous precursor-filled liposome is popped by the sonic pulse from theultrasound, the prodrug encapsulated by the liposome will then beexposed to the serum. The ester linkage is then cleaved by esterases inthe serum, thereby generating the drug.

Similarly, ultrasound may be utilized not only to rupture the gaseousprecursor-filled liposome, but also to cause thermal effects which mayincrease the rate of the chemical cleavage and the release of the activedrug from the prodrug.

The liposomes may also be designed so that there is a symmetric or anasymmetric distribution of the drug both inside and outside of theliposome.

The particular chemical structure of the therapeutics may be selected ormodified to achieve desired solubility such that the therapeutic mayeither be encapsulated within the internal gaseous precursor-filledspace of the liposome, attached to the liposome or enmeshed in theliposome. The surface-bound therapeutic may bear one or more acyl chainssuch that, when the bubble is popped or heated or ruptured viacavitation, the acylated therapeutic may then leave the surface and/orthe therapeutic may be cleaved from the acyl chains chemical group.Similarly, other therapeutics may be formulated with a hydrophobic groupwhich is aromatic or sterol in structure to incorporate into the surfaceof the liposome.

The present invention is further described in the following examples,which illustrate the preparation and testing of the gaseousprecursor-filled liposomes. Examples 1-5, and 22-24 are actual; Examples6-21 are prophetic. The following examples should not be construed aslimiting the scope of the appended claims.

EXAMPLE 1 Preparation of Gas-Filled Lipid Spheres from Perfluorobutane

Gaseous precursor-containing liposomes were prepared usingperfluorobutane (Pfaltz and Bauer, Waterbury, Conn.) as follows: A 5 mLsolution of lipid, 5 mg per ml, lipid=87 mole percent DPPC, 8 molepercent DPPE-PEG 5,000, 5 mole percent dipalmitoylphosphatidic acid (alllipids from Avanti Polar Lipids, Alabaster, Ala.), in 8:1:1 normalsaline:glycerol:propylene glycol, was placed in a glass bottle with arubber stopper (volume of bottle=15.8 ml). Air was evacuated from thebottle using a vacuum pump, Model Welch 2-Stage DirecTorr Pump (VWRScientific, Cerritos, Calif.) by connecting the hose to the bottlethrough a 18 gauge needle which perforated the rubber stopper. Afterremoving the gas via vacuum, perfluorobutane was placed in the stopperedbottle via another 18 gauge needle connected to tubing attached to thecanister of perfluorobutane. This process was repeated 5 times such thatany traces of air were removed from the stoppered bottle and the spaceabove the lipid solution was completely filled with perfluorobutane. Thepressure inside the glass bottle was equilibrated to ambient pressure byallowing the 18 gauge needle to vent for a moment or two before removingthe 18 gauge needle from the stopper. After filling the bottle withperfluorobutane the bottle was secured to the arms of a Wig-L-Bug™(Crescent Dental Mfg. Co., Lyons, Ill.) using rubber bands to fasten thebottle. The bottle was then shaken by the Wig-L-Bug™ for 60 seconds. Afrothy suspension of foam resulted and it was noted that it took severalminutes for any appreciable separation of the foam layer from the clearsolution at the bottom. After shaking, the volume of the materialincreased from 5 cc to about 12 cc, suggesting that the liposomesentrapped about 7 cc of the perfluorocarbon gaseous precursor. Thematerial was sized using an Accusizer (Model 770, Particle SizingSystem, Santa Barbara, Calif.) and also examined by a light polarizingmicroscope (Nikon TMS, Nikon) at 150 x magnification power. Theliposomes appeared as rather large spherical structure with meandiameter of about 20 to 50 microns. A portion of these liposomes wasthen injected via a syringe through a Costar filter (Syrfil 800938,Costar, Pleasanton, Calif.) with pore sizes of 8 microns. The liposomeswere again examined via light microscope and the Accusizer System. Themean size of the liposomes was about 3 microns and the volume weightedmean was about 7 microns. Greater that 99.9 percent of the liposomeswere under 11 microns in size. The above experiment exhibits the use ofa gaseous precursor gas, perfluorobutane, can be used to make verydesirable sized liposomes by a process of shaking and filtration.

The above was substantially repeated except that after filling thebottle with perfluorobutane at room temperature the bottle was thentransferred to a freezer and the material subjected to a temperature of-20° C. At this temperature the perfluorobutane became liquid. Becauseof the glycerol and propylene glycol, the lipid solution did not freeze.The bottle was quickly transferred to the Wig-L-Bug™ and subjected toshaking as described above for three cycles, one minute each, at roomtemperature. During this time the contents of the bottle equilibrated toroom temperature and was noted to be slightly warm to the touchsecondary to the energy imparted through shaking by the Wig-L-Bug™. Atthe end of vortexing a large volume of foam was again noted similar tothat described above. The resulting liposomes were again studied bylight microscopy and Accusizer. A portion was then subjected tofiltration sizing through an 8 micron filter as described above andagain studied by microscope and Accusizer. The results from sizing weresubstantially the same as with the gaseous precursor as described above.

Imaging was performed in a New Zealand White rabbit weighing about 3.5kg. The animal was sedated with rabbit mix (Xylene 10 mg/ml; Ketamine100 mg/ml and Acepromazine 20 mg/ml) and scanned with an AcousticImaging, Model No. 7200, clinical ultrasound machine, scanning thekidney by color doppler with a 7.5 MHz transducer. Simultaneously whilethe kidney was scanned the rabbits heart was also scanned using a secondAcoustic Imaging ultrasound machine, model n. 5200, with a 7.5 MHztransducer for grey scale imaging of the heart. Injection of theperfluorobutane-filled liposomes was administered via ear vein through asyringe fitted with a 8 micron filter (see above). After injection of0.5 cc (0.15 cc per kg) of liposomes containing the gaseous precursorperfluorobutane, dramatic and sustained enhancement of the kidney wasobserved for over 30 minutes. This was shown as brilliant color withinthe renal parenchyma reflecting increased signal within the renalarcuate arteries and microcirculation. The simultaneous imaging of theheart demonstrated shadowing for the first several minutes whichprecluded visualization of the heart, i.e. the reflections were sostrong the ultrasound beam was completely reflected and absorbed. Afterseveral minutes, however, brilliant and sustained ventricular and bloodpool enhancement was observed which also persisted for more than 50minutes. Images were also obtained of the liver using the grey scaleultrasound machine. These showed parenchymal and vascular enhancement atthe same time as the cardiac and blood pool enhancement.

In summary, this experiment demonstrates how liposomes can be used toentrap a gaseous precursor and create very stable liposomes of definedand ideal size. The invention has vast potential as an ultrasoundcontrast agent and for drug delivery. Because the liposomes are sostable they will pass through the target tissue (a tumor for example)via the circulation. Energy can then be focused on the target tissueusing ultrasound, microwave radiofrequency or magnetic fields to pop theliposomes and perform local drug delivery.

EXAMPLE 2 Preparation of Gaseous Precursors Via Microfluidization

Gaseous precursor-filled lipid bilayers were prepared as in Example 1except, after addition of the gaseous precursor, the contents weremicrofluidized through six passes on a Microfluidics microfluidizer(Microfluidics Inc., Newton, Mass.). The stroke pressure ranged between10,000 and 20,000 psi. Continuing with the preparation as per Example 1,produced gas-filled lipid bilayers with gaseous precursor encapsulated.

EXAMPLE 3 Formulation of Gas-Filled Lipid Bilayers Using PhosphatidicAcid and Dipalmitoyphosphatidylcholine

Gas-filled lipid bilayers were prepared as set forth in Example 1 exceptfor the fact that DPPC was used in combination with 5 mole %phosphatidic acid (Avanti Polar Lipids, Alabaster, Ala.). Formulation ofgas-filled lipid bilayers resulted in an increase in solubility asexemplified by a decrease in the amount of lipid particulate in thelower aqueous vehicle layer. Resultant sizing appeared to decrease theoverall mean size vs. DPPC alone to less than 40 um.

EXAMPLE 4

Formulation of Gas-Filled Lipid Bilayers Using Phosphatidic Acid,Dipalmitoylphosphatidylethanolamine-PEG 5,000 andDipalmitoylphosphatidylcholine

Perfluorobutane encapsulated lipid bilayers were formed as discussed inExample 3 except that the lipid formulation contained 82%dipalmitoylphosphatidylcholine, 10 mole % dipalmitoylphosphatidic acid,and 8 mole % dipalmitoylphosphatidylethanolamine-PEG 5,000 (Avanti PolarLipids, Alabaster, Ala.) in a vehicle consisting of 8:1:1 (v:v:v) normalsaline:propylene glycol:glycerol, yielding a foam and a lower vehiclelayer that was predominantly devoid of any particulate. Variations ofthis vehicle yielded varying degrees of clarity to the lower vehiclelayer. The formulation was prepared identically as in Example 3 to yieldgas-filled lipid bilayers containing perfluorobutane. Prior tofiltration, the gas-filled microspheres were sized on a Particle SizingSYstems Model 770 optical sizer (Particle Sizing Systems, Santa Barbara,Calif.). Sizing resulted in 99% of all particles residing below 34 μm.The resultant product ws then filtered through an 8 μm filter to yieldmicrospheres of uniform size. Sizing of the subsequent microspheresresulted in 99.5% of all particles residing below 10 μm. This productwas used in the in vivo experiments in Example 1.

It is noted that the vehicle was altered with other viscosity modifiersand solubilizers in varying proportions which resulted in greater orlesser degrees of clarity and particulate. Amongst a variety of lipidsand lipid analogs used in combination, it was subsequently found thatthe introduction of DPPE-PEG lipid significantly improved the sizedistribution and apparent stability of the gas-filled lipid bilayers.

EXAMPLE 4A Binding of DNA by Gas-Filled Lipid Bilayers

Binding of DNA by liposomes containing phosphatidic acid and gaseousprecursor and gas containing liposomes. A 7 mM solution ofdistearoyl-sn-glycerophospate (DSPA) (Avanti Polar Lipids, Alabaster,Ala.) was suspended in normal saline and vortexed at 50° C. The materialwas allowed to cool to room temperature. 40 micrograms of pBR322 plasmidDNA (International Biotechnologies, Inc., New Haven, Conn.) was added tothe lipid solution and shaken gently. The solution was centrifuged for10 minutes in a Beckman TJ-6 Centrifuge (Beckman, Fullerton, Calif.).The supernatant and the precipitate were assayed for DNA content using aHoefer TKO-100 DNA Fluorometer (Hoefer, San Francisco, Calif.). Thismethod only detects double stranded DNA as it uses an intercalating dye,Hoechst 33258 which is DNA specific. It was found that the negativelycharged liposomes, or lipids with a net negative charge, prepared withphosphatidic acid surprisingly bound the DNA. This experiment wasrepeated using neutral liposomes composed of DPPC as a control. Noappreciable amount of DNA was detected with the DPPC liposomes. Theexperiment was repeated using gas-filled liposomes prepared from an87:8:5 mole percent of DPPC to DPPE-PEG 500 to DPPA mixture of lipids ina microsphere. Again, the DNA bound to the gas-filled liposomescontaining dipalmitoylphosphatidic acid.

EXAMPLE 5 Microemulsification of Precursor

A Microfluidizer (Microfluidics, Newton, Mass.) was placed in a coldroom at -20° C. A stoppered glass flask containing a head space of 35 ccof perfluorobutane and 25 cc of lipid solution was taken into the coldroom. The lipid solution contained an 83:8:5 molar ratio ofDPPC:DPPE+PEG 5,000:DPPA in 8:1:1 phosphate buffered saline (pH7.4):glycerol:propylene glycol. The solution did not freeze in the coldroom but the perfluorobutane became liquid.

The suspension of lipids and liquid gaseous precursor was then placedinto the chamber of the Microfluidizer and subjected to 20 passes at16,000 psi. Limited size vesicles, having a size of about 30 nm to about50 nm, resulted. Upon warming to room temperature, stabilizedmicrospheres of about 10 microns resulted.

EXAMPLE 6 Preparation of Gaseous Precursor-filled Liposomes

Fifty mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (MW: 734.05,powder, Lot No. 160pc-183) (Avanti-Polar Lipids, Alabaster, Ala.) isweighed and hydrated with 5.0 ml of saline solution (0.9% NaCl) orphosphate buffered saline (0.8% sodium chloride, 0.02% potassiumchloride, 0.115% dibasic sodium phosphate and 0.02% monobasic potassiumphosphate, pH adjusted to 7.4) in a centrifuge tube. To this suspensionis added 165 μL mL⁻¹ of 2-methyl-2-butene. The hydrated suspension isthen shaken on a vortex machine (Scientific Industries, Bohemia, N.Y.)for 10 minutes at an instrument setting of 6.5. A total volume of 12 mlis then noted. The saline solution is expected to decrease from 5.0 mlto about 4 ml.

The gaseous precursor-filled liposomes made via this new method are thensized by optical microscopy. It will be determined that the largest sizeof the liposomes ranged from about 50 to about 60 μm and the smallestsize detected is about 8 μm. The average size ranges from about 15 toabout 20 μm.

The gaseous precursor-filled liposomes are then filtered through a 10 or12 μm "NUCLEPORE" membrane using a Swin-Lok Filter Holder, (NucleporeFiltration Products, Costar Corp., Cambridge, Mass.) and a 20 cc syringe(Becton Dickinsion & Co., Rutherford, N.J.). The membrane is a 10 or 12μm "NUCLEPORE" membrane (Nuclepore Filtration Products, Costar Corp.,Cambridge, Mass.). The 10.0 μm filter is placed in the Swin-Lok FilterHolder and the cap tightened down securely. The liposome solution isshaken up and transferred to the 20 cc syringe via an 18 gauge needle.Approximately 12 ml of liposome solution is placed into the syringe, andthe syringe is screwed onto the Swin-Lok Filter Holder. The syringe andthe filter holder assembly are inverted so that the larger of thegaseous precursor-filled liposomes vesicles could rise to the top. Thenthe syringe is gently pushed up and the gaseous precursor-filledliposomes are filtered in this manner.

The survival rate (the amount of the gaseous precursor-filled liposomesthat are retained after the extrusion process) of the gaseousprecursor-filled liposomes after the extrusion through the 10.0 μmfilter is about 83-92%. Before hand extrusion, the volume of foam isabout 12 ml and the volume of aqueous solution is about 4 ml. After handextrusion, the volume of foam is about 10-11 ml and the volume ofaqueous solution is about 4 ml.

The optical microscope is used again to determine the size distributionof the extruded gaseous precursor-filled liposomes. It will bedetermined that the largest size of the liposomes range from about 25 toabout 30 μm and the smallest size detected is about 5 μm. The averagesize range is from about 8 to about 15 μm.

It is found that after filtering, greater than 90% of the gaseousprecursor-filled liposomes are smaller than 15 μm.

EXAMPLE 7 Preparation of Gaseous Precursor-Filled LiposomesIncorporating Lyophilization

Fifty mg of 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine, (MW: 734.05,powder) (Avanti-Polar Lipids, Alabaster, Ala.) is weighed and placedinto a centrifuge tube. The lipid is then hydrated with 5.0 ml of salinesolution (0.9% NaCl). To this suspension is added 165 μL mL⁻¹ of2-methyl-2-butene. The lipid is then vortexed for 10 minutes at aninstrument setting of 6.5. After vortexing, the entire solution isfrozen in liquid nitrogen. Then the sample is put on the lyophilizer forfreeze drying. The sample is kept on the lyophilizer for 18 hours. Thedried lipid is taken off the lyophilizer and rehydrated in 5 ml ofsaline solution and vortexed for ten minutes at a setting of 6.5. Asmall sample of this solution is pipetted onto a slide and the solutionis viewed under a microscope. The size of the gaseous precursor-filledliposomes will then be determined. It will be determined that thelargest size of the liposomes is about 60 μm and the smallest sizedetected is about 20 μm. The average size range is from about 30 toabout 40 μm.

EXAMPLE 8 Example of Gaseous Precursor-filled Liposome Preparation Abovethe Phase Transition Temperature of the Lipid

Fifty mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (MW: 734.05,powder) (Avanti-Polar Lipids, Alabaster, Ala.) is weighed and placedinto a centrifuge tube. To this suspension is added 165 μL mL⁻¹ of2-methyl-2-butene. Approximately two feet of latex tubing (0.25 in.inner diameter) is wrapped around a conical centrifuge tube in acoil-like fashion. The latex tubing is then fastened down to thecentrifuge tube with electrical tape. The latex tubing is then connectedto a constant temperature circulation bath (VWR Scientific Model 1131).The temperature of the bath is set to 60° C. and the circulation ofwater is set to high speed to circulate through the tubing. Athermometer is placed in the lipid solution and found to be between 42°C. and 50° C.

The lipid solution is vortexed for a period of 10 minutes at a vortexinstrument setting of 6.5. It will be noted that very little foaming ofthe lipid (phase transition temp.=41° C.) and that the suspension didnot appreciably form gaseous precursor-filled liposomes. Opticalmicroscopy revealed large lipidic particles in the solution. The numberof gaseous precursor-filled liposomes that form at this temperature isless than 3% of the number that form at a temperature below the phasetransition temperature. The solution is allowed to sit for 15 minutesuntil the solution temperature equilibrated to room temperature (25°C.). The solution is then vortexed for a duration of 10 minutes. After10 minutes, it will be noted that gaseous precursor-filled liposomesformed.

EXAMPLE 9 Preparation of Gaseous Precursor-filled LiposomesIncorporating a Freeze-Thaw Procedure

50 mg of 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (MW: 734.05,powder) (Avanti-Polar Lipids, Alabaster, Ala.) is weighed and placedinto a centrifuge tube. The lipid is then hydrated with 5.0 ml of 0.9%NaCl added. To this suspension is added 165 μL mL⁻¹ of2-methyl-2-butene. The aqueous lipid solution is vortexed for 10 minutesat an instrument setting of 6.5. After vortexing, the entire solution isfrozen in liquid nitrogen. The entire solution is then thawed in a waterbath at room temperature (25° C.). The freeze thaw procedure is thenrepeated eight times. The hydrated suspension is then vortexed for 10minutes at an instrument setting of 6.5. Gaseous precursor-filledliposomes are then detected as described in Example 6.

EXAMPLE 10 Preparation of Gaseous Precursor-Filled Liposomes with anEmulsifying Agent (Sodium Lauryl Sulfate)

Two centrifuge tubes are prepared, each having 50 mg of DPPC. 1 mol %(˜0.2 mg of Duponol C lot No. 2832) of sodium lauryl sulfate is added toone of the centrifguge tubes, and the other tube receives 10 mol % (2.0mg of Duponol C lot No. 2832). Five ml of 0.9% NaCl is added to bothcentrifuge tubes. 165 μL mL⁻¹ of 2-methyl-2-butene is added to bothtubes. Both of the tubes are frozen in liquid nitrogen and lyophilizedfor approximately 16 hours. Both samples are removed from thelyophilizer and 5 ml of saline is added to both of the tubes. Both ofthe tubes are vortexed at position 6.5 for 10 minutes.

It will be determined that the largest size of the gaseousprecursor-filled liposomes with 1 mol % of sodium lauryl sulfate isabout 75 μm and the smallest size detected is about 6 μm. The averagesize range is from about 15 to about 40 μm. It will be determined thatthe largest size of the gaseous precursor-filled liposomes with 10 mol %of sodium lauryl sulfate is about 90 μm and the smallest size detectedis about 6 μm. The average size range is from about 15 to about 35 μm.

The volume of foam in the solution containing gaseous precursor-filledliposomes with 1 mol % sodium lauryl sulfate is about 15 ml and thevolume of aqueous solution is about 3-4 ml. The volume of foam in thesolution containing gaseous precursor-filled liposomes with 10 mol %sodium lauryl sulfate is also about 15 ml and the volume of aqueoussolution is about 3-4 ml.

EXAMPLE 11 Determination of Whether Gaseous Precursor-Filled LiposomesCan Be Generated by Sonication

50 mg of lipid, 1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine(Avanti-Polar Lipids, Alabaster, Ala.), is weighed out and hydrated with5 ml of 0.9% NaCl. To this suspension is added 165 μL/mL⁻¹ of2-methyl-2-butene. Instead of vortexing, the aqueous solution issonicated using a Heat Systems Sonicator Ultrasonic Processor XL (HeatSystems, Inc., Farmingdale, N.Y.) Model XL 2020. The sonicator, with afrequency of 20 KHz, is set to continuous wave, at position 4 on theknob of the sonicator. A micro tip is used to sonicate for 10 minutes ata temperature of 4° C. Following sonication, the temperature isincreased to 40° C. and the solution is viewed under an opticalmicroscope. There will be evidence of gaseous precursor-filled liposomeshaving been produced.

Next, the above is repeated with sonication at a temperature of 50° C.and 165 μL mL⁻¹ of 2-methyl 2-butene is added. The micro tip of thesonicator is removed and replaced with the end cap that is supplied withthe sonicator. Another solution (50 mg of lipid per 5 ml of saline) isprepared and sonicated with this tip. After 10 minutes, the solution isviewed under the microscope. The production of gas-filled liposomes withsonication above the temperature of the transition of the gas resultedin a lower yield of gas-filled lipid spheres.

EXAMPLE 12 Determination of Concentration Effects on GaseousPrecursor-Filled Liposome Production

This example determined whether a lower concentration limit of the lipidhalts the production of gaseous precursor-filled liposomes. Ten mg of1,2-Dipalmitoyl-Sn-Glycero-3-Phosphocholine (Avanti-Polar Lipids,Alabaster, Ala.) is added to 10 ml of saline. To this suspension isadded 165 μL/mL⁻¹ of 2-methyl-2-butene. The lipid/saline/gas precursorsolution is vortexed at position 6.5 for 10 minutes. The solution isviewed under an optical microscope for sizing. It will be determinedthat the largest size of the liposomes ranges from about 30 to about 45μm and the smallest size detected is about 7 μm. The average size rangeis from about 30 to about 45 μm.

It appears that the gaseous precursor-filled liposomes are more fragileas they appear to burst more rapidly than previously shown. Thus, itappears that concentration of the lipid is a factor in the generationand stability of gaseous precursor-filled liposomes.

EXAMPLE 13 Cascade Filtration

Unfiltered gaseous precursor-filled liposomes may be drawn into a 50 mlsyringe and passed through a cascade of a "NUCLEPORE" 10 μm filter and 8μm filter that are a minimum of 150 μm apart (FIGS. 3 and 4).Alternatively, for example, the sample may be filtered through a stackof 10 μm and 8 μm filters that are immediately adjacent to each other.Gaseous precursor-filled liposomes are passed through the filters at apressure whereby the flow rate is 2.0 ml min⁻¹. The subsequentlyfiltered gaseous precursor-filled liposomes are then measured for yieldof gaseous precursor-filled lipid liposomes which results in a volume of80-90% of the unfiltered volume.

The resulting gaseous precursor-filled liposomes are sized by fourdifferent methods to determine their size and distribution. Sizing isperformed on a Particle Sizing Systems Model 770 Optical Sizing unit, aZeiss Axioplan optical microscope interfaced to image processingsoftware manufactured by Universal Imaging, and a Coulter Counter(Coulter Electronics Limited, Luton, Beds., England). As seen in FIGS. 5and 6, the size of the gaseous precursor-filled liposomes are moreuniformly distributed around 8-10 μm as compared to the unfilteredgaseous precursor-filled liposomes. Thus, it can be seen that thefiltered gaseous precursor-filled liposomes are of much more uniformsize.

EXAMPLE 14 Preparation of Filtered DPPC Suspension

250 mg DPPC (dipalmitoylphosphatidylcholine) and 10 ml of 0.9% NaCl areadded to a 50 ml Falcon centrifuge tube (Becton-Dickinson, Lincoln Park,N.J.) and maintained at an ambient temperature (approx. 20° C.). To thissuspension is added 165 μL/mL⁻¹ of 2-methyl-2-butene. The suspension isthen extruded through a 1 μm Nuclepore (Costar, Pleasanton, Calif.)polycarbonate membrane under nitrogen pressure. The resultant suspensionis sized on a Particle Sizing Systems (Santa Barbara, Calif.) Model 370laser light scattering sizer. All lipid particles are 1 μm or smaller inmean outside diameter.

In addition, the same amount of DPPC/gas precursor suspension is passedfive times through a Microfluidics™ (Microfluidics Corporation, Newton,Mass.) microfluidizer at 18,000 p.s.i. The suspension, which becomesless murky, is sized on a Particle Sizing Systems (Santa Barbara,Calif.) Sub Micron Particle Sizer Model 370 laser light scattering sizerwhere it is found that the size is uniformly less than 1 μm. Theparticle size of microfluidized suspensions is known to remain stable upto six months.

EXAMPLE 15 Preparation of Filtered DSPC Suspension

100 mg DSPC (distearoylphosphatidylcholine) and 10 ml of 0.9% NaCl areadded to a 50 ml Falcon centrifuge tube (Becton-Dickinson, Lincoln Park,N.J.). To this suspension is added 165 μL/mL⁻¹ of 2-methyl-2-butene. Thesuspension is then extruded through a 1 μm "NUCLEPORE" (Costar,Pleasanton, Calif.) polycarbonate membrane under nitrogen pressure at300-800 p.s.i. The resultant suspension is sized on a Particle SizingSystems (Santa Barbara, Calif.) Sub Micron Particle Sizer Model 370laser light scattering sizer. It will be found that all particles are 1μm or smaller in size.

In addition, the same amount of DPPC/gas precursor suspension is passedfive times through a Microfluidics™ (Microfluidics Corporation, Newton,Mass.), microfluidizer at 18,000 p.s.i. The resultant suspension, whichis less murky, is sized on a Sub Micron Particle Sizer Systems Model 370laser light scattering sizer and it is found that the size is uniformlyless than 1 μm.

EXAMPLE 16 Sterilization of Filtered Lipid Suspensions by Autoclaving

The previously sized suspensions of DPPC/gas precursor and DSPC/gasprecursor of Examples 9 and 10 are subjected to autoclaving for twentyminutes on a Barnstead Model C57835 autoclave (Barnstead/Thermolyne,Dubuque, Iowa) and then subjected to shaking. A filtration step may beperformed immediately prior to use through an in line filter. Also, thegaseous precursor may be autoclaved before sizing and shaking.

After equilibration to room temperature (approx. 20° C.), the sterilesuspension is used for gaseous precursor instillation.

EXAMPLE 17 Gaseous Precursor Instillation of Filtered, Autoclaved LipidsVia Vortexing

10 ml of a solution of 1,2-dipalmitoylphosphatidylcholine at 25 mg/ml in0.9% NaCl, which had previously been extruded through a 1 μm filter andautoclaved for twenty minutes, is added to a Falcon 50 ml centrifugetube (Becton-Dickinson, Lincoln Park, N.J.). To this suspension is added165 μL/mL⁻¹ of 2-methyl-2-butene. After equilibration of the lipidsuspension to room temperature (approximately 20° C.), the liquid isvortexed on a VWR Genie-2 (120 V, 0.5 amp, 60 Hz.) (ScientificIndustries, Inc., Bohemia, N.Y.) for 10 minutes or until a time that thetotal volume of gaseous precursor-filled liposomes is at least double ortriple the volume of the original aqueous lipid solution. The solutionat the bottom of the tube is almost totally devoid of anhydrousparticulate lipid, and a large volume of foam containing gaseousprecursor-filled liposomes results. Thus, prior autoclaving does notaffect the ability of the lipid suspension to form gaseousprecursor-filled liposomes. Autoclaving does not change the size of theliposomes, and it does not decrease the ability of the lipid suspensionsto form gaseous precursor-filled liposomes.

EXAMPLE 18 Gaseous Precursor Instillation of Filtered, Autoclaved LipidsVia Shaking on Shaker Table

10 ml of a solution of 1,2-dipalmitoylphosphatidylcholine at 25 mg/ml in0.9% NaCl, which has previously been extruded through a 1 μm filter andautoclaved for twenty minutes, is added to a Falcon 50 ml centrifugetube (Becton-Dickinson, Lincoln Park, N.J.). To this suspension is added165 μL/mL⁻¹ of perfluoropentane (PCR Research Chemicals, Gainesville,Fla.). After equilibration of the lipid suspension to room temperature(approximately 20° C.), the tube is then placed upright on a VWRScientific Orbital shaker (VWR Scientific, Cerritos, Calif.) and shakenat 300 r.p.m. for 30 minutes. The resultant agitation on the shakertable results in the production of gaseous precursor-filled liposomes.

EXAMPLE 18A

The above experiment may be performed replacing perfluoropentane withsulfur hexafluoride, hexafluoropropylene, bromochlorofluoromethane,octafluoropropane, 1,1 dichloro, fluoro ethane, hexafluoroethane,hexafluoro-2-butyne, perfluoropentane, perfluorobutane,octafluoro-2-butene or hexafluorobuta-1,3-diene oroctafluorocyclopentene, all with the production of gaseous precursorfilled liposomes.

EXAMPLE 19 Gaseous Precursor Instillation of Filtered, Autoclaved LipidsVia Shaking on Shaker Table Via Shaking on Paint Mixer

10 ml of a solution of 1,2-dipalmitoylphosphatidylcholine at 25 mg/ml in0.9% NaCl, which has previously been extruded through a 1 μm filter andautoclaved for twenty minutes, is added to a Falcon 50 ml centrifugetube (Becton-Dickinson, Lincoln Park, N.J.). To this suspension is added165 μL/mL⁻¹ of 2-methyl-2-butene. After equilibration of the lipidsuspension to room temperature (approximately 20° C.), the tube isimmobilized inside a 1 gallon empty household paint container andsubsequently placed in a mechanical paint mixer employing a gyratingmotion for 15 minutes. After vigorous mixing, the centrifuge tube isremoved, and it is noted that gaseous precursor-filled liposomes form.

EXAMPLE 20 Gaseous Precursor Instillation of Filtered, Autoclaved LipidsVia Shaking by Hand

10 ml of a solution of 1,2-dipalmitoylphosphatidylcholine at 25 mg/ml in0.9% NaCl, which had previously been extruded through a 1 μm nucleporefilter and autoclaved for twenty minutes, is added to a Falcon 50 mlcentrifuge tube (Becton-Dickinson, Lincoln Park, N.J.). To thissuspension is added 165 μL/mL⁻¹ of 2-methyl-2-butene. Afterequilibration of the lipid suspension to room temperature (approximately20° C.), the tube is shaken forcefully by hand for ten minutes. Uponceasing agitation, gaseous precursor-filled liposomes form.

EXAMPLE 21 Sizing Filtration of Autoclaved Gaseous Precursor-FilledLiposomes Via Cascade or Stacked Filters

Gaseous precursor-filled liposomes are produced from DPPC as describedin Example 17. The resultant unfiltered liposomes are drawn into a 50 mlsyringe and passed through a cascade filter system consisting of a"NUCLEPORE" (Costar, Pleasanton, Calif.) 10 μm filter followed by an 8μm filter spaced a minimum of 150 μm apart. In addition, on a separatesample, a stacked 10 μm and 8 μm filtration assembly is used, with thetwo filters adjacent to one another. Gaseous precursor-filled liposomesare passed through the filters at a pressure such that they are filtereda rate of 2.0 ml/min. The filtered gaseous precursor-filled liposomesyields a volume of 80-90% of the unfiltered volume.

The resultant gaseous precursor-filled liposomes are sized by fourdifferent methods to determine their size distribution. Sizing isperformed on a Particle Sizing Systems (Santa Barbara, Calif.) Model 770Optical Sizing unit, and a Zeiss (Oberkochen, Germany) Axioplan opticalmicroscope interfaced to image processing software (Universal Imaging,West Chester, Pa.) and a Coulter Counter (Coulter Electronics Limited,Luton, Beds., England). As illustrated in FIG. 8, the size of thegaseous precursor-filled liposomes is more uniformly distributed around8-10 μm as compared to the unfiltered gaseous precursor-filledliposomes.

EXAMPLE 22 Efficient Production of Gas-Precursor Filled Lipid Spheres

The same procedure as in Example 6 is performed except that the shakerused is a Crescent "Wig-L-Bug™ (Crescent Manufacturing Dental Co.,Lyons, Ill.). The formulation is then agitated for 60 seconds instead ofthe usual 5 minutes to 10 minutes as described previously. Gas-filledlipid spheres are produced.

EXAMPLE 23

100 μL of perfluoropentane (bp 29.5° C., PCR Research Chemicals,Gainesville, Fla.) was added to a 5 mg/mL lipid suspension and vortexedon a Genie II mixer (Scientific Industries, Inc., Bohemia, N.Y.) at roomtemperature at power setting of 6.5. A Richmar (Richmar Industries,Inola, Okla.) 1 MHz therapeutic ultrasound device was then used toperform hyperthermia, elevating the temperature to above 42° C. asmeasured by a thermometer. Upon reaching the phase transitiontemperature, gas microspheres were noted. A simultaneous scanning wasperformed with a diagnostic ultrasound (Acoustic Imaging, Phoenix,Ariz.). Acoustic signals from the gas microspheres could also bevisualized on the clinical diagnostic ultrasound.

The same exeriment was conducted with octafluorocyclopentene (bp 27° C.,PCR Research Chemicals, Gainesville, Fla.).

EXAMPLE 24

An experiment identical to Example 23 was performed where the suspensionwas vortexed and injected into a Harlan-Sprague Dawley rat, 300 grams,previously given a C5A tumor cell line in the left femoral region. ARichmar 1 MHz therapeutic ultrasound was then placed over the tumorregion and an adriamycin embedded lipid suspension injectedintravenously. The therapeutic ultrasound was then placed on acontinuous wave (100% duty cycle) setting and the tumor heated. A secondrat, having a C5A tumor cell line in the left femoral region, was givenan identical dose of the adriamycin emulsion, however, no ultrasound wasutilized in this animal. Within three weeks it was noted that the tumor,compared to the control without the use of ultrasound, was noticeablysmaller.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. Gaseous precursor-filled liposomes prepared by agel state shaking gaseous precursor instillation method, said methodcomprising adding a lipid to an aqueous solution or suspension, shakingsaid lipid-containing aqueous solution or suspension in the presence ofa temperature activated gaseous precursor at a temperature below the gelstate to liquid crystalline state phase transition temperature of thelipid, wherein said gaseous precursor is a fluorine-containing gaseousprecursor, said fluorine-containing gaseous precursor selected from thegroup consisting of perfluorocarbons, fluorohydrocarbons, and sulfurhexafluoride, wherein upon activation of said gaseous precursor to a gassaid liposomes have at least about a 90% volume of gas in the interiorthereof.
 2. Gas-filled liposomes prepared by a gel state shaking gasinstillation method, said liposomes comprising a lipid and afluorine-containing gas, said fluorine-containing gas selected from thegroup consisting of perfluorocarbons, fluorohydrocarbons, and sulfurhexafluoride, said liposomes prepared by a method comprising adding alipid to an aqueous solution or suspension, shaking saidlipid-containing aqueous solution or suspension in the presence of atemperature activated gaseous precursor at a temperature below the gelstate to liquid crystalline state phase transition temperature of thelipid, said liposomes having at least about a 90% volume of gas in theinterior thereof.
 3. The liposomes of claim 2 wherein said lipid isselected from the group consisting of fatty acids; lysolipids;sphingolipids; glycolipids; glucolipids; sulfatides; glycosphingolipids;lipids with ether and ester-linked fatty acids, polymerized lipids,phospholipids with short chain fatty acids of 6-8 carbons in length, andsynthetic phospholipids with asymmetric acyl chains.
 4. The liposomes ofclaim 3 wherein said polymer is between 400 and 200,000 molecularweight.
 5. The liposomes of claim 3 wherein said polymer is between1,000 and 20,000 molecular weight.
 6. The liposomes of claim 3 whereinsaid polymer is between 2,000 and 8,000 molecular weight.
 7. Theliposomes of claim 3 wherein said lipid bearing a covalently boundpolymer comprises compounds of the formula XCHY--(CH₂)n--O--(CH₂)n--YCHXwherein X is an alcohol group, Y is OH or an alkyl group and n is 0 to10,000.
 8. The liposomes of claim 3 wherein said polymer is selectedfrom the group consisting of polyethyleneglycol, polyvinylpyrrolidone,polyvinylalcohol and polypropyleneglycol.
 9. The liposomes of claim 3wherein said polymer is polyethyleneglycol.
 10. The liposomes of claim 3wherein said lipid comprises from about 1 mole % to about 20 mole %. 11.The gas-filled liposomes of claim 2 wherein said fluorine-containing gasis selected from the group consisting of fluorine gas, 1-fluorobutane,hexafluoro acetone, tetrafluoroallene, boron trifluoride,1,2,3-trichloro, 2-fluoro-1,3-butadiene, hexafluoro-1,3-butadiene,1-fluoro-butane, 1,2,3-trichloro, 2-fluoro-1,3-butadiene,hexafluoro-1,3-butadiene, 1-fluoro-butane, decafluoro butane,perfluoro-1-butene, perfluoro-1-butene, perfluoro-2-butene,2-chloro-1,1,1,4,4,4-hexafluoro-butyne, perfluoro-2-butyne,octafluoro-cyclobutane, perfluoro-cyclobutene, perfluoroethane,perfluoropropane, perfluorobutane, perfluoropentane, perfluorohexane,1,1,1-trifluorodiazoethane, hexafluoro-dimethyl amine,perfluorodimethylamine, 4-methyl,1,1,1,2-tetrafluoro ethane,1,1,1-trifluoroethane, 1,1,2,2-tetrafluoroethane,1,1,2-trichloro-1,2,2-trifluoroethane, 1,1-dichloro-1,2,2,2-tetrafluoroethane, 1,2-difluoro ethane, 1-chloro-1,1,2,2,2-pentafluoro ethane,2-chloro, 1,1-difluoroethane, 1-chloro-1,1,2,2-tetrafluoro ethane,2-chloro, 1,1-difluoroethane, chloropentafluoro ethane,dichlorotrifluoroethane, fluoro-ethane, hexafluoro-ethane,nitro-pentafluoro ethane, nitroso-pentafluoro ethane, perfluoro ethane,perfluoro ethylamine, 1,1-dichloro-1,2-difluoro ethylene, 1,2-difluoroethylene, methane-sulfonyl chloride-trifluoro, methanesulfonylfluoride-trifluoro, methane-(pentafluorothio)trifluoro, methane-bromodifluoro nitroso, methane-bromo fluoro, methane-bromo chloro-fluoro,methanebromo-trifluoro, methane-chloro difluoro nitro, methanechlorofluoro, methane-chloro trifluoro, methane-chloro-difluoro, methanedibromo difluoro, methane-dichloro difluoro, methane-dichloro-fluoro,methanedifluoro, methane-difluoro-iodo, methane-fluoro,methane-iodo-trifluoro, methane-nitro-trifluoro,methane-nitroso-trifluoro, methane-tetrafluoro, methane-trichlorofluoro,methane-trifluoro, methanesulfenylchloride-trifluoro, pentane-perfluoro,1-pentane-perfluoro, propane-1, 1, 1, 2, 2, 3-hexafluoro, propane-2,2difluoro, propane-heptafluoro-1-nitro, propane-heptafluoro-1-nitroso,propane-perfluoro, propyl-1,1,1,2,3,3-hexafluoro-2,3 dichloro,propylene-3-fluoro, propylene-perfluoro, propyne-3,3,3-trifluoro,styrene-3-fluoro, sulfur hexafluoride, sulfur (di)-decafluoro,trifluoroacetonitrile, trifluoromethyl peroxide, trifluoromethylsulfide, tungsten hexafluoride, pentafluoro octadecyl iodide,perfluorooctylbromide, perfluorodecalin, perfluorododecalin,perfluorooctyliodide, perfluorotripropylamine, perfluorotributylamine,hexafluoropropylene, bromochlorofluoromethane, octafluoropropane, 1,1dichloro, fluoro ethane, hexa- fluoroethane, hexafluoro-2-butyne,perfluoropentane, perfluorobutane, octafluoro-2-butene,hexafluorobuta-1,3-diene, and octafluorocyclopentene.
 12. The gas-filledliposomes of claim 11 wherein said fluorine-containing gas is selectedfrom the group consisting of fluorine gas, perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane,perfluorohexane, sulfur hexafluoride, hexafluoropropylene,octafluoropropane, perfluorocyclobutane, octafluorocyclopentene,dodecafluoropentane, and octafluorocyclobutane.
 13. The liposomes ofclaim 12 wherein said fluorine-containing gas is selected from the groupconsisting of perfluoromethane, perfluoroethane, perfluoropropane,perfluorobutane, perfluorocyclobutane, and sulfur hexafluoride.
 14. Theliposomes of claim 13 wherein said fluorine-containing gas is selectedfrom the group consisting of perfluoropropane, perfluorocyclobutane, andperfluorobutane.
 15. The liposomes of claim 14 wherein saidfluorine-containing gas is perfluoropropane.
 16. The liposomes of claim2 wherein said fluorine-containing gas is a perfluorocarbon gas.
 17. Theliposomes of claim 16 wherein said perfluorocarbon gas is selected fromthe group consisting of perfluoromethane, perfluoroethane,perfluoropropane, perfluorobutane, and perfluorocyclobutane.
 18. Theliposomes of claim 17 wherein said perfluorocarbon gas is selected fromthe group consisting of perfluoropropane, perfluorocyclobutane, andperfluorobutane.
 19. The liposomes of claim 18 wherein saidperfluorocarbon gas is perfluoropropane.
 20. The gaseousprecursor-filled liposomes of claim 1 for use with a nebulizer, saidliposomes targeted to the lung.
 21. The liposomes of claim 2 whereinsaid lipid is selected from the group consisting of phosphatidylcholine,dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine,dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine,dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,distearoylphosphatidylcholine, phosphatidylethanolamine,dioleoylphosphatidylethanolamine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol, sphingomyelin, gangliosideGM1, ganglioside GM2, phosphatidic acid, palmitic acid, stearic acid,arachidonic acid, oleic acid, lipids bearing polyethyleneglycol, chitin,hyaluronic acid or polyvinylpyrrolidone, cholesterol, cholesterolsulfate, cholesterol hemisuccinate, tocopherol hemisuccinate, diacetylphosphate, stearylamine, cardiolipin,6-(5-cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside,digalactosyldiglyceride,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside,6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-α-D-mannopyranoside,12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoicacid, N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino)octadecanoyl]-2-aminopalmitic acid,cholesteryl)4'-trimethyl-ammonio)butanoate,N-succinyldioleoylphosphatidylethanol-amine,1,2-dioleoyl-sn-glycerol,1,2-dipalmitoyl-sn-3-succinylglycerol,1,3-dipalmitoyl-2-succinylglycerol,1-hexadecyl-2-palmitoylglycerophosphoethanolamine,palmitoylhomocysteine, lauryltrimethylammonium bromide,cetyltrimethylammonium bromide, myristyltrimethylammonium bromide,alkyldimethylbenzylammonium chloride, benzyldimethyldodecylammoniumbromide, benzyldimethylhexadecylammonium bromide,benzyldimethyltetradecylammonium bromide, cetyldimethylethylammoniumbromide, cetylpyridinium bromide, pentafluoro octadecyl iodide,perfluorooctylbromide, perfluorodecalin, perfluorododecalin,perfluorooctyliodide, perfluorotripropylamine, andperfluorotributylamine.